Christophe Bailly
email: christophe.bailly@sorbonne-universite.fr

The signalling roles of reactive oxygen species in the regulation of seed germination and dormancy

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The signalling roles of reactive oxygen species in the regulation of seed germination and dormancy

Introduction

Oxygen supports aerobic life, but can also give rise to the reactive oxygen species (ROS). These include free radicals, which contain an unpaired electron such as singlet oxygen ( 1 O 2 ), superoxide ( O ⋅ -2 ) or hydroxyl radical ( • OH) and hydrogen peroxide (H 2 O 2 ), which is not a free radical. Hydrogen peroxide is considered as the main ROS involved in cellular signalling as it is rather stable (lifetime of milliseconds) and is capable of crossing biological membranes [START_REF] Petrov | Hydrogen peroxide-a central hub for information flow in plant cells[END_REF]. Although ROS have distinct chemical properties, they are all highly reactive and can cause damage to other biomolecules. Oxidative stress is well documented in plants since it generally accompanies biotic and abiotic stresses in higher plants and can ultimately lead to cell death [START_REF] Demidchik | Mechanisms of oxidative stress in plants: From classical chemistry to cell biology[END_REF]. In contrast, ROS are also recognized as key signalling intermediates involved in a wide range of plant responses to the environment and as regulators of plant development [START_REF] Mittler | ROS Are Good[END_REF] [START_REF] Waszczak | Reactive Oxygen Species in Plant Signaling[END_REF].

More than a decade ago it was proposed that ROS were involved in the control of seed germination and dormancy release [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF] [START_REF] Bailly | From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology[END_REF]. At this time, ROS were principally associated with oxidative damage and loss of viability during seed ageing. The involvement of ROS homeostasis in regulating seed germination and dormancy came in 2007 when Oracz et al. [START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF] demonstrated that sunflower seed dormancy alleviation during dry storage was associated with ROS accumulation and protein carbonylation. Since this time, ROS progressively emerged as essential components of the germination process leading to the concept of "oxidative window for germination", which restricts the occurrence of the cellular events associated with germination to a critical range of ROS level, enclosed by lower and higher limits [START_REF] Bailly | From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology[END_REF]. There is now further evidence showing that ROS homeostasis is critical for germination, and this is examined in this review article.

Seeds are spectacular and intriguing organisms. They are at the core of plant kingdom because they retain the genetic information of higher plant species, which survival and dissemination depends on seed longevity and successful germination and establishment of a novel plant. Nevertheless, the particularities of seeds, when compared to whole plant systems, has to be considered when investigating the role of ROS in their germination. First, the socalled "orthodox seeds" desiccate on the mother plant at the end of their developmental program, reaching very low moisture content [START_REF] Bewley | Seed Germination and Dormancy[END_REF]. This desiccation phase can be considered as an extreme drought stress associated with ROS generation and oxidative stress, from which developing seeds are protected through enzymatic and non-enzymatic mechanisms [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF]. As a consequence, at shedding, the status of dry mature orthodox seeds is largely oxidized. The physiology of seeds evolves during subsequent dry storage. In particular this period may be necessary to alleviate dormancy, a blockage of germination in apparently favourable environmental conditions, in a process termed after-ripening [START_REF] Finch-Savage | Seed dormancy and the control of germination[END_REF]. Desiccated seeds are a quiescent state where no metabolism can be detected, but non-enzymatic ROS generation has been frequently evidenced in these conditions [START_REF] Bailly | From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology[END_REF] [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF]. Then, during imbibition, seed water uptake allows metabolism resumption and subsequently radicle protrusion, the first visible sign of the completion of germination, if the environmental conditions are permissive, i.e. if water potential, temperature, oxygen and light conditions are appropriate, and if seeds are non-dormant [START_REF] Finch-Savage | Seed dormancy and the control of germination[END_REF]. The recovery of an active metabolism is associated with enzymatic and regulated production of ROS. In contrast to whole plants, imbibed seeds are very sensitive to small variations of environmental factors. This sensitivity is an adaptive trait which allows germination to proceed only when environmental conditions are appropriate for subsequent seedling development, thus increasing the probabilities of survival of the resulting seedlings.

For example, a difference of few degrees Celsius can impose a block on seed germination, a phenomena which is emphasized in dormant seeds. This suggests that environmental signals have to be accurately transmitted to seeds and translated into endogenous signals regulating germination. In this review we propose that ROS are very candidates for the fine-tuning of the timing of germination. Lastly, the relationship of ROS with other cell signalling pathways, i.e. plant hormones, is often different in seeds than in other plant systems. For example, abscisic acid (ABA) and ROS interact synergistically in response to plant abiotic stress such as drought, in particular for regulating stomata opening [START_REF] Mittler | The Roles of ROS and ABA in Systemic Acquired Acclimation[END_REF], but in seeds ROS and ABA are generally antagonists. This particularity has also to be considered when studying ROS signalling in seeds.

The objective of this review is to provide a critical view of the signalling role of ROS in seed germination and dormancy, taking into account the recent findings in both germination and ROS signalling mechanisms.

Evidence for a role of ROS in germination and dormancy

Table 1 ([7], ) displays some of the works that have been published within the last ten years dealing with the signalling role of ROS in seed germination and dormancy. In this table we have distinguished beneficial and detrimental effects of ROS accumulation on completion of germination or dormancy release. It appears clearly that most of these works have evidenced a positive role of ROS in these processes, whatever the species. These former studies have shown that controlled ROS accumulation during seed imbibition is a prerequisite to radicle elongation, as well as dormancy alleviation, either by cold stratification or afterripening. In this context many attempts have been performed in order to investigate whether ROS homeostasis crosstalk with the major hormonal regulators of germination and dormancy, i.e. ABA, gibberellins (GA) or ethylene, and the major findings will be discussed below. In contrast, another set of studies has also pointed out the deleterious role of ROS in the completion of seed germination. In this context, their accumulation during seed imbibition triggers oxidative stress and slows down or inhibits germination. Such an effect has been evidenced when seeds were germinated in stressful conditions either caused by drought, salt stress or toxic compounds in the imbibition medium, for example (Table 1).

These studies confirm and validate the concept of the oxidative window for germination [START_REF] Bailly | From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology[END_REF]. In this concept the ability of a seed to germinate is directly depended on ROS homeostasis: seed germination is likely to occur only when the seed ROS content is enclosed within values that allows ROS signalling but not ROS damage. In contrast germination is thus prevented when the amount of ROS is too low or too elevated. Although there are no more doubts about the involvement of ROS in germination, many questions about their production and mode of action remain unsolved.

ROS production in seeds

The orthodox seeds, which undergo a dramatic desiccation phase at the end of their developmental program on planta, can survive for years in a resting and anhydrobiotic state.

However, in such a desiccated state, major physiological changes such as dormancy alleviation (so called dry after-ripening) or ageing are likely to occur. Seeds can also withstand an abrupt and invasive water uptake during their imbibition which will drop their moisture content from values below 8-10 % dry weight basis (dwb) to values higher than 50 % dwb at the onset of radicle protrusion. This plasticity of life has marked consequence on metabolism since it is almost at a standstill in dry mature seeds and is progressively reactivated during imbibition. Thus this almost unique feature (with other anhydrobiotes) of metabolic variation is in consequence associated with marked changes in ROS production.

ROS metabolism and production in dry seeds are rarely investigated maybe because it remains technically challenging (most methods implies to use water, and then to modify the initial seed status). The "fuel" for producing ROS in anhydrobiosis is oxygen that exists in its ground state ( 3 O2) with two unpaired electrons with parallel spins [START_REF] Halliwell | Role of free radicals and catalytic metal ions in human disease: an overview[END_REF] and its reduction gives rise to the various forms of ROS. In dry seeds oxygen is likely to be present in void spaces which altogether constitute an air space network [START_REF] Cloetens | Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network[END_REF]. In addition, seed desiccation on the mother plant generates ROS thus creating an oxidative environment within tissues of the mature seed [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF]. Providing oxygen is present, chemical (non-enzymatic) reactions of ROS production can thus proceed during seed storage. Lipids in particular are very prone to oxidation at low moisture content and can serve as a source of free radicals. This process is well described in food science and lipid oxidation has a U-shaped relationship to water activity (aw), increasing at very low moisture contents. Increasing moisture content in zone I of water sorption isotherm decreases lipid oxidation by eliminating pores and decreasing oxygen exposure of lipids to oxygen. It was proposed that solubility or sorption of molecular oxygen in dehydrated foods is lowered as water content increases and, thus, the reaction rate is lowered by decreasing the effective oxygen concentration [START_REF] Karel | Lipid Oxidation, Secondary Reactions, and Water Activity of Foods[END_REF] [START_REF] Barden | Lipid Oxidation in Low-moisture Food: A Review[END_REF]. Moreover in low moisture systems peroxidation of lipids may lead to reactions of proteins with lipid hydroperoxides, free radicals and peroxide breakdown products [START_REF] Karel | Lipid Oxidation, Secondary Reactions, and Water Activity of Foods[END_REF].

The occurrence of ROS production during seed dry after-ripening has been demonstrated either directly by measuring change in ROS content (eg [START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF]) or by following oxidation of biomolecules within seeds ( [START_REF] Bazin | Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening[END_REF][63] [START_REF] Morscher | Glutathione redox state, tocochromanols, fatty acids, antioxidant enzymes and protein carbonylation in sunflower seed embryos associated with after-ripening and ageing[END_REF]). For example, after-ripening of sunflower embryonic axes was accompanied by a shift in the thiol-based cellular redox environment towards more oxidizing conditions [START_REF] Morscher | Glutathione redox state, tocochromanols, fatty acids, antioxidant enzymes and protein carbonylation in sunflower seed embryos associated with after-ripening and ageing[END_REF] and by oxidation of proteins [START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF] and mRNAs [START_REF] Bazin | Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening[END_REF]. In barley, the ROS content in the embryo was not affected by after-ripening, while the antioxidant glutathione (GSH) was gradually converted to glutathione disulphide (GSSG) [START_REF] Bahin | Crosstalk between reactive oxygen species and hormonal signalling pathways regulates grain dormancy in barley[END_REF]. The key role of oxygen in seed dormancy release during dry storage has been experimentally demonstrated recently by Buijs et al. [START_REF] Buijs | Seed dormancy release accelerated by elevated partial pressure of oxygen is associated with DOG loci[END_REF] who increased the internal partial pressure of oxygen within Arabidopsis seeds to accelerate dormancy release. Indirect evidence were also given by Bazin et al. [START_REF] Bazin | Role of relative humidity, temperature, and water status in dormancy alleviation of sunflower seeds during dry after-ripening[END_REF] and Basbouss-Serhal et al. [START_REF] Basbouss-Serhal | Fluctuation of Arabidopsis seed dormancy with relative humidity and temperature during dry storage[END_REF] who studied dormancy alleviation of sunflower and Arabidopsis seeds, respectively, in a wide range of temperature and relative humidities. Both demonstrated that at low moisture content (below 7 % dwb) dormancy alleviation was associated with negative energy activation, as determined by Arrhenius plots. Negative activation energies suggest that the kinetics of a biological process increases when temperature decreases and it reveals non-enzymatic processes, related to ROS production and oxidation mechanisms [START_REF] Bazin | Role of relative humidity, temperature, and water status in dormancy alleviation of sunflower seeds during dry after-ripening[END_REF] thus confirming the measured changes in ROS content during dry after-ripening.

ROS production in imbibed seeds is better characterized and reviewed previously [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF].

The studies of Bazin et al. [START_REF] Bazin | Role of relative humidity, temperature, and water status in dormancy alleviation of sunflower seeds during dry after-ripening[END_REF] and Basbouss-Serhal et al. [START_REF] Basbouss-Serhal | Fluctuation of Arabidopsis seed dormancy with relative humidity and temperature during dry storage[END_REF] demonstrated that metabolic activity in seeds could resume when moisture content reached values as low as 10 % dwb in sunflower and ca. 13 % dwb in Arabidopsis. Kibinza et al. [67] also demonstrated that respiration in sunflower embryonic axes became effective at 10 % moisture content.

Therefore even a limited water uptake can trigger ROS production trough metabolic activities. Commonly detected ROS in imbibed seeds are superoxide, hydrogen peroxide and hydroxyl radical (see studies shown in Table 1). The absence of functional chloroplast in seeds does not favour production of singlet oxygen, although it has already been detected in germinating seeds but it was considered as being arising from lipid peroxidation [START_REF] Chen | Rapid determination of rice seed vigour by spontaneous chemiluminescence and singlet oxygen generation during early imbibition[END_REF]. In imbibed seeds ROS are probably mainly produced by mitochondria, since 2-3% of oxygen used by this organelle is supposed to generates superoxide anion at complexes I and II of the electron transfer chain, then giving hydrogen peroxide [START_REF] Puntarulo | Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination[END_REF]. H 2 O 2 can further react through Fenton reaction with reduced Fe 2+ and Cu 2+ to produce • OH [START_REF] Richards | The hydroxyl radical in plants: from seed to seed[END_REF]. Whether mitochondrial ROS plays a role in germination, their production should be regulated, and this mechanism is far from being known. Recently Ma et al. [START_REF] Ma | Mitochondrial small heat shock protein mediates seed germination via thermal sensing[END_REF] demonstrated that a mitochondrial matrixlocalized heat shock protein could induce reactive oxygen species in a temperature dependent manner which strengthen the role of ROS as environmental sensors in the germination process. The other candidates often mentioned in the context of regulation of germination by ROS are NADPH oxidases. NADPH oxidases (NOXs), also known as respiratory burst oxidase homologues (RBOHs), are certainly the most-studied ROS-producing enzymes [START_REF] Marino | A burst of plant NADPH oxidases[END_REF].

They transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygen to produce superoxide in the apoplast which is rapidly dismutated to H 2 O 2 . The activity of NADPH oxidases has been frequently proposed to be involved in ROS production during seed imbibition (eg [START_REF] Mueller | In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth[END_REF] [START_REF] Leymarie | Role of Reactive Oxygen Species in the Regulation of Arabidopsis Seed Dormancy[END_REF][START_REF] Kai | Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination[END_REF][START_REF] Ishibashi | A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination[END_REF][START_REF] Li | Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination[END_REF]) and, similarly to other plant systems, NOXs could play a role in sensing environmental conditions favourable for germination.

Attention has also to be paid to the diverse sources of apoplastic ROS production, since apoplastic ROS can play a critical role in cell wall weakening which precedes cell elongation. The apoplastic copper-containing amine oxidases and polyamine oxidases catalyse deamination of di-and polyamines and produce H 2 O 2 [START_REF] Kärkönen | Reactive oxygen species in cell wall metabolism and development in plants[END_REF]. Oxalate oxidases (germins and germin-like proteins) catalyse the oxidation of oxalate to CO 2 and H 2 O 2 [START_REF] Hu | Overexpression of a Gene Encoding Hydrogen Peroxide-Generating Oxalate Oxidase Evokes Defense Responses in Sunflower[END_REF].

Peroxidases and quinone reductase also generate ROS [START_REF] Liszkay | Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth[END_REF] [START_REF] Schopfer | Naphthoquinone-Dependent Generation of Superoxide Radicals by Quinone Reductase Isolated from the Plasma Membrane of Soybean[END_REF]. Most of these enzymes have been frequently cited as being involved in the late phase of germination, at the onset of radicle protrusion.

ROS content does not only depend on ROS production systems but is also regulated by the efficiency of enzymatic and non-enzymatic antioxidant mechanisms (already reviewed in seeds by [START_REF] Bailly | Active oxygen species and antioxidants in seed biology[END_REF]). Changes in activities of superoxide dismutase, catalase and enzymes of the ascorbate-glutathione cycle have been widely investigated in the context of seed germination. To understand the role of these systems in the regulation of seed germination, one nevertheless has to distinguish between seed germination under favourable conditions from germination under stressful conditions. In the first case it is difficult to get a clear picture on the role of these systems prior to radicle protrusion. In some case it has been demonstrated that activities of antioxidant enzymes increased during imbibition of germinating seeds [START_REF] Bykova | Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control[END_REF][START_REF] Krasuska | Nitric oxide and hydrogen cyanide as regulating factors of enzymatic antioxidant system in germinating apple embryos[END_REF][START_REF] Cembrowska-Lech | Germination induction of dormant Avena fatua caryopses by KAR(1) and GA(3) involving the control of reactive oxygen species (H2O2 and O-2(center dot-)) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers[END_REF][START_REF] Anand | Hydrogen peroxide signaling integrates with phytohormones during the germination of magnetoprimed tomato seeds[END_REF] while in other cases these activities decreased [START_REF] Ma | Cell signaling mechanisms and metabolic regulation of germination and dormancy in barley seeds[END_REF]. Moreover, in most cases, activation of antioxidant systems is a late event in the germination process. We suggest that activation of these systems only occur when ROS level exceed a certain value, in order to maintain ROS homeostasis within the oxidative window for germination, which means that in permissive conditions of germination the level of ROS is rather under the control of ROS generating mechanisms. When seeds are germinated in non-optimal conditions there is a positive correlation between ROS scavenging ability through antioxidant systems and germination rate. For example, among only recent studies, seed antioxidant enzyme activities have been demonstrated to increase in response to high temperature and drought stress [START_REF] Liu | High temperature and drought stress cause abscisic acid and reactive oxygen species accumulation and suppress seed germination growth in rice[END_REF][START_REF] Zhou | Signaling Molecule Hydrogen Sulfide Improves Seed Germination and Seedling Growth of Maize (Zea mays L.) Under High Temperature by Inducing Antioxidant System and Osmolyte Biosynthesis[END_REF], low temperature [START_REF] Luo | Associating transcriptional regulation for rapid germination of rapeseed (Brassica napus L.) under low temperature stress through weighted gene co-expression network analysis[END_REF], salt stress [START_REF] Guzman-Murillo | Germination and ROS detoxification in bell pepper (Capsicum annuum L.) under NaCl stress and treatment with microalgae extracts[END_REF][START_REF] Ahammed | Epigallocatechin-3-Gallate Alleviates Salinity-Retarded Seed Germination and Oxidative Stress in Tomato[END_REF] or mutagen agents [START_REF] Zhang | Mutagen-induced phytotoxicity in maize seed germination is dependent on ROS scavenging capacity[END_REF]. In those cases, the activation of antioxidant enzymes is necessary to prevent excessive ROS accumulation and related oxidative damage. In contrast, in the context of seed dormancy, activities of antioxidant enzymes have often been shown to be lower, or even impaired, in non-dormant seeds than in dormant seeds, whatever the species [START_REF] Oracz | The Mechanisms Involved in Seed Dormancy Alleviation by Hydrogen Cyanide Unravel the Role of Reactive Oxygen Species as Key Factors of Cellular Signaling during Germination[END_REF][START_REF] Leymarie | Role of Reactive Oxygen Species in the Regulation of Arabidopsis Seed Dormancy[END_REF][START_REF] Ishibashi | The Interrelationship between Abscisic Acid and Reactive Oxygen Species Plays a Key Role in Barley Seed Dormancy and Germination[END_REF][START_REF] Zhang | Carbonylated protein changes between active germinated embryos and quiescent embryos give insights into rice seed germination regulation[END_REF][START_REF] Bi | Arabidopsis ABI5 plays a role in regulating ROS homeostasis by activating CATALASE 1 transcription in seed germination[END_REF]. In this context, their reduced efficiency can participate, in association with increase ROS production mechanisms, in the increased accumulation of ROS involved in dormancy release. Altogether the available data suggest that ROS can translate environmental cues to signals in seeds, as suggested by the concept of oxidative window, and that the fine-tuning of their production helps the seed to make a decision when to germinate. This most likely occurs through the interaction with hormone signalling pathways.

There remain many unanswered questions relating to the regulation of ROS production in time and space. For example, it has been proposed recently that ROS signal could autopropagate in plants leading to the concept of ROS wave, where ROS could diffuse from cell to cell via RBOHD [START_REF] Miller | The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli[END_REF]. With regards to the functional morphology of seeds, where the elongation zone of the embryonic axis is located behind the meristematic area [START_REF] Sliwinska | Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl[END_REF], we can hypothesize that localized ROS production is important for germination control. We have examined this hypothesis in germinating seeds, using cell imaging of ROS production (Figure 1). We show that there is a dynamic production of ROS along the embryonic axis in germinating seeds of Arabidopsis (Figure 1). At an early timepoint of germination (6 h) ROS are mostly located in the meristem area of the radicle (Figures 1A,1D) and further imbibition progressively relocalizes the maximum area of ROS production in the elongating zone of the hypocotyl (Figuress 1B-C and 1E-F), thus evidencing a "developmental wave" of ROS production during the germination process. This suggests that specific cell territories, could sense environmental cues, i.e. the meristem area, and initiate a ROS signal propagating along the embryonic axis and ultimately lead to cell elongation in the hypocotyl area. This finding has to be considered with regards to the spatiotemporal cell expansion mechanisms, related to ABA and GA metabolism and signalling, involved the seed to seedling transition in Arabidopsis [START_REF] Stamm | The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination[END_REF] and who share some similarities with spatiotemporal ROS production.

Similarly, at the cellular level, during the germination process, it has also been demonstrated that ROS were first localized in the cytoplasm, then in the nucleus and finally in the cell wall at the time cells elongate [START_REF] Leymarie | Role of Reactive Oxygen Species in the Regulation of Arabidopsis Seed Dormancy[END_REF]. ROS may therefore have distinct roles during the germination process, ranging from interaction with cytoplasmic signalling pathways (early germination), oxidative regulation of gene expression within the nucleus (mid-germination) and cell wall weakening (late germination). Thus a better understanding of role(s) of ROS in germination will require paying attention to the spatial regulation of their homeostasis at the whole organ and at the subcellular level.

ROS signalling is mediated by direct oxidation of biomolecules

Considering the effect of ROS as signals in the germination process requires to pay attention to their downstream effects. Interestingly the strong reactivity of ROS makes possible their direct oxidative action on various cellular components and this can in turn modify cell functioning and in consequence whole seed physiology.

Proteins and nucleic acids can be easily oxidized by ROS. The role of these processes have been described previously in details [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF] and will be only briefly evoked here. The amino acids Cys and Met are very sensitive to oxidation. The oxidation of thiol to disulfide, is a known redox regulation mechanism, the reduced form being regenerated by the glutaredoxin and thioredoxin (Trx) systems. Various studies, reviewed in [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF], have shown that germination might be associated with protein thiol oxidation, probably in balance with reduction of low molecular weight thiol disulfides [START_REF] Bykova | Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control[END_REF][START_REF] Gerna | Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth[END_REF][START_REF] Gerna | Redox poise and metabolite changes in bread wheat seeds are advanced by priming with hot steam[END_REF]. Besides oxidation of sulfurcontaining amino acids, carbonylation is the most common oxidative and non-reversible protein modification. It can result in loss of function and even degradation of the carbonylated proteins. Extensive protein carbonylation has been evidenced during germination of Arabidopsis, pea and rice seeds [START_REF] Job | Patterns of protein oxidation in Arabidopsis seeds and during germination[END_REF][START_REF] Barba-Espin | Role of thioproline on seed germination: Interaction ROS-ABA and effects on antioxidative metabolism[END_REF][START_REF] Zhang | Carbonylated protein changes between active germinated embryos and quiescent embryos give insights into rice seed germination regulation[END_REF], and it was mostly directed towards reserve proteins. These authors proposed that carbonylation can be important for mobilization of seed storage proteins because it increases protein susceptibility towards proteolytic cleavage by 20S proteasome. In addition it was shown that carbonylation was not randomly distributed among proteins which suggests that oxidation of specific proteins is likely to play a role in the completion of germination. This idea was confirmed by Oracz et al. [START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF] who compared carbonylation patterns of dormant and non-dormant sunflower seeds and who showed that carbonylation was directed towards negative regulators of germination. In rice, Zhang et al. [START_REF] Zhang | Carbonylated protein changes between active germinated embryos and quiescent embryos give insights into rice seed germination regulation[END_REF] showed that HSP and LEA, 2 known positive regulators of dormancy, became carbonylated during germination.

RNAs are also very sensitive to non-enzymatic oxidation [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF]. The presence of oxidized bases in mRNAs causes translation errors and produces truncated proteins [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF].

Bazin et al. [START_REF] Bazin | Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening[END_REF] and Gao et al. [START_REF] Gao | Integrated analysis of seed proteome and mRNA oxidation reveals distinct posttranscriptional features regulating dormancy in wheat (Triticum aestivum L.)[END_REF] demonstrated that specific oxidation of a subset of transcripts was a prerequisite for dormancy release of sunflower and wheat seeds, respectively. It seems therefore that one of the role of ROS is to trigger direct oxidation of negative regulators of germination, as proposed by [START_REF] El-Maarouf-Bouteau | Role of protein and mRNA oxidation in seed dormancy and germination[END_REF].

ROS can directly interact with cell wall polysaccharides and promote cell elongation in germinating seeds. The hydroxyl radical, in particular, is considered as a plant cell wall loosening agent which can directly cleave wall polysaccharides [START_REF] Fry | Oxidative scission of plant cell wall polysaccharides by ascorbateinduced hydroxyl radicals[END_REF]. . OH can be formed either enzymatically (see previous section) or by Fenton reaction involving a transition metal [START_REF] Kärkönen | Reactive oxygen species in cell wall metabolism and development in plants[END_REF]. In endospermic seeds, it has been demonstrated that endosperm cap weakening by ROS was an important process for allowing the elongating radicle to penetrate through the endosperm cap [START_REF] Mueller | In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth[END_REF][START_REF] Zhang | Involvement of reactive oxygen species in endosperm cap weakening and embryo elongation growth during lettuce seed germination[END_REF][START_REF] Steinbrecher | Tissue and cellular mechanics of seeds[END_REF]. Interestingly this production is inhibited by ABA and promoted by GA and ethylene [START_REF] Mueller | In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth[END_REF], thus underlying again the close connection between ROS and hormone signalling pathways. ROS have also been shown to be produced in cell walls of growing embryos, at the time of radicle protrusion [START_REF] Steinbrecher | Tissue and cellular mechanics of seeds[END_REF], including for non-endospermic seed species [START_REF] Kranner | Extracellular production of reactive oxygen species during seed germination and early seedling growth in Pisum sativum[END_REF]. Depending on the species, it has been proposed that cell wall peroxidases, NADPH oxidases or polyamine oxidases were involved in ROS production in the apoplast of elongating cells of the embryonic axis [START_REF] Mueller | In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth[END_REF][START_REF] Linkies | Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum[END_REF][START_REF] Zhang | Involvement of reactive oxygen species in endosperm cap weakening and embryo elongation growth during lettuce seed germination[END_REF][START_REF] Chen | Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds[END_REF][START_REF] Li | Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination[END_REF][START_REF] Singh | Early axis growth during seed germination is gravitropic and mediated by ROS and calcium[END_REF]. Independently of the mechanisms of production, there is growing evidence showing that apoplastic ROS production is necessary for initiating cell elongation in the growing radicle. Such feature should also been considered with the recent findings of Stamm et al. [START_REF] Stamm | The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination[END_REF] who highlighted the role of GA in the spatiotemporal cell expansion mechanisms in Arabidopsis.

ROS crosstalk with germination signalling pathways

Signalling ROS effect on germination cannot be considered as whole but must be regarded as a part of a complex signalling network. Germination and dormancy being tightly regulated by hormones [START_REF] Finch-Savage | Seed dormancy and the control of germination[END_REF] it is therefore highly relevant to estimate the role of ROS in the context of hormone signalling and metabolism. The relationship between ROS and the hormones involved in germination, i.e. mostly ABA and GA, is now well documented but needs to be considered under various angles. In one way, increase in ROS content, which is beneficial for germination, is associated with alteration of synthesis and signalling of ABA, the hormone which represses germination. It has been demonstrated that H 2 O 2 accumulation in germinating seeds was associated with ABA degradation probably trough an activation of ABA-8-hydroxylase, an ABA catalytic enzyme [START_REF] Liu | H2O2 mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination[END_REF][START_REF] Ishibashi | The Interrelationship between Abscisic Acid and Reactive Oxygen Species Plays a Key Role in Barley Seed Dormancy and Germination[END_REF][START_REF] Ishibashi | A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination[END_REF][START_REF] Li | Reactive Oxygen Species and Gibberellin Acid Mutual Induction to Regulate Tobacco Seed Germination[END_REF][START_REF] Anand | Hydrogen peroxide signaling integrates with phytohormones during the germination of magnetoprimed tomato seeds[END_REF] even though a direct oxidation of ABA cannot be excluded [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Cutler | Formation and breakdown of ABA[END_REF][START_REF] Zhou | A New Abscisic Acid Catabolic Pathway[END_REF]. ABA being regulating germination by its antagonistic interaction with GA it is worth noting that ROS have been reported to stimulate GA biosynthesis through a transcriptional effect [START_REF] Bahin | Crosstalk between reactive oxygen species and hormonal signalling pathways regulates grain dormancy in barley[END_REF][START_REF] Kai | Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination[END_REF][START_REF] Ishibashi | A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination[END_REF][START_REF] Li | Reactive Oxygen Species and Gibberellin Acid Mutual Induction to Regulate Tobacco Seed Germination[END_REF][START_REF] Anand | Hydrogen peroxide signaling integrates with phytohormones during the germination of magnetoprimed tomato seeds[END_REF]. In consequence ROS homeostasis has a direct effect on the hormonal balance ABA/GA in the favour of GA which in term induces germination. To get a complete view of the relationship ROS/hormones it is also necessary to consider the effect of hormones on ROS production and scavenging. Direct imbibition of seeds with ABA, the use of mutants altered in ABA synthesis or signalling and the comparison of dormant to non-dormant seeds have shown that ABA repress ROS production in seeds [START_REF] Lariguet | Identification of a hydrogen peroxide signalling pathway in the control of lightdependent germination in Arabidopsis[END_REF][START_REF] Bi | Arabidopsis ABI5 plays a role in regulating ROS homeostasis by activating CATALASE 1 transcription in seed germination[END_REF][START_REF] Bi | Arabidopsis ABI5 plays a role in regulating ROS homeostasis by activating CATALASE 1 transcription in seed germination[END_REF][START_REF] Amooaghaie | Triangular interplay between ROS, ABA and GA in dormancy alleviation of Bunium persicum seeds by cold stratification[END_REF][START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF]. Conversely, GA treatment has been shown to induce ROS production [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Cembrowska-Lech | Germination induction of dormant Avena fatua caryopses by KAR(1) and GA(3) involving the control of reactive oxygen species (H2O2 and O-2(center dot-)) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers[END_REF]. The relationship between others hormones which are putatively playing a role in germination and dormancy is less documented. Ethylene is a gaseous plant hormone which stimulates the germination of dormant seeds of many species [START_REF] Corbineau | Ethylene, a key factor in the regulation of seed dormancy[END_REF]. A synergic relationship between ROS and ethylene has been demonstrated in sunflower seeds, with ethylene triggering ROS production [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Oracz | The Mechanisms Involved in Seed Dormancy Alleviation by Hydrogen Cyanide Unravel the Role of Reactive Oxygen Species as Key Factors of Cellular Signaling during Germination[END_REF][START_REF] Corbineau | Ethylene, a key factor in the regulation of seed dormancy[END_REF][START_REF] Oracz | Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway[END_REF]. Even though other plant hormones are likely to lay role in germination, their possible interaction with ROS is not yet understood.

In plants, ROS, and mostly H 2 O 2 , have been reported to interact with other signalling pathways [START_REF] Petrov | Hydrogen peroxide-a central hub for information flow in plant cells[END_REF][START_REF] Waszczak | Reactive Oxygen Species in Plant Signaling[END_REF][START_REF] Singh | Reactive Oxygen Species (ROS): Beneficial Companions of Plants' Developmental Processes[END_REF]. For example, mitogen-activated protein kinases (MAPK) can be activated However all these interactions are poorly described in the context of seed germination, and among them only the relationship between ROS and NO has retain some attention some years ago [106].

Changes in ROS homeostasis can induce changes in seed gene expression. In plants the effect of ROS on transcriptomes have been studied using ROS-generating treatments or mutants altered in ROS scavenging/production [107]. A global meta-analysis of microarray data obtained in such conditions allowed to define transcriptional footprints and to design a so-called ROS wheel, in which co-regulated genes in response to ROS are clustered together [108]. This approach is relevant as it reveals oxidative components in a biological process using transcriptomic data. As an example, we have performed this analysis using the data obtained by Basbouss-Serhal et al. [109]. In this study the authors have identified a set of transcripts which became specifically associated with polysomes (i.e. the translatome) during imbibition of dormant and non-dormant Arabidopsis seeds. Such transcripts are likely to be translated and the corresponding proteins may then play a role in germination or dormancy.

We have used the ROS wheel to investigate the ROS signature at the level of the translatome during seed imbibition (Table 2). The ROS wheel reveals the occurrence of ROS metabolism in seed germination, either in dormant or non-dormant seeds. Many transcripts range within the cluster III-High Light Early, especially in dormant seeds. This former cluster consists of transcription profiles triggered by high light exposures [108] and its over representation can appear surprising, but this is the largest cluster of the ROS wheel (more than 400 genes) and it also includes genes of response to heat or to H 2 O 2 , for example. Nevertheless the ROS wheel also allows the discrimination of dormant from non-dormant seeds. It is worth noting that it reveals the occurrence of retrograde signaling in non-dormant seeds only, a process in which ROS production in either chloroplasts or mitochondria control nuclear gene expression. This is in agreement with the recent findings of Ma et al. [START_REF] Ma | Mitochondrial small heat shock protein mediates seed germination via thermal sensing[END_REF] who showed the role of mitochondrial ROS production in the germination process. The extended use of ROS footprints should help to better assess the involvement of ROS in seed germination.

The effects of ROS on transcriptional reprogramming during seed germination has been poorly investigated to date. In sunflower, a microarray analysis showed that treatment of dormant seeds by methylviologen, a ROS generating compound inducing dormancy release, modified the expression of 120 genes. Most of the identified transcripts were related to cell signalling components [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF]. Interestingly genes down-regulated in methylviologen-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level.

Conclusions

The beneficial role of ROS in the regulation of seed germination is increasingly well established with the series of studies reviewed here. This relationship is not always easy to address because the same ROS have versatile effects and can either favour or repress germination, which are simultaneously balanced with the adverse effects these molecules can have on cells. Figure 2 summarizes the main findings and hypotheses presented in this review. We propose that spatiotemporal regulation of ROS production in embryonic axis of imbibed seeds controls the ability to germinate. The 3 steps model presented Figure 2 suggests that sensing of environmental conditions is detected in the meristem area and that controlled production of ROS triggers germination mechanisms, i.e. activation of GA signalling pathway and oxidation of negative regulators of germination. At this early time point ROS homeostasis mostly depend upon production sources. The localized increases in ROS generates an unknown signal that can propagate along the embryonic axis and trigger cell elongation in the hypocotyl area at the onset of radicle protrusion. At late imbibition time, antioxidant systems participate to the control of ROS homeostasis and ROS are generated within the apoplast to participate to cell wall loosening. Further studies are required to better understand how environmental factors can fine tune ROS production, how the oxidative signal is translated at the subcellular level and how it can propagate from cell to cell. It is clear that ROS and hormones act in concert to regulate seed germination but the bases of this crosstalk are far from being understood. It will be of a particular interest to consider spatiotemporal ROS production with regards to the recently evidenced spatiotemporal mode of action of hormones in the germination process [START_REF] Stamm | The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination[END_REF]110]. confocal microscopy using Leica TCS SP5 confocal microscope, as described in [START_REF] Leymarie | Role of Reactive Oxygen Species in the Regulation of Arabidopsis Seed Dormancy[END_REF] and [START_REF] Ishibashi | Regulation of soybean seed germination through ethylene production in response to reactive oxygen species[END_REF]. Fluorescence intensity was estimated using Image-J software. 1. At the beginning of imbibition, environmental conditions are perceived by the imbibed seed at the meristematic region where a limited ROS production occurs through mitochondria electron transfer chain and NADPH oxidase activity. It induces the oxidation of negative regulators of germination, activation of gibberellin (GA) signalling and inactivation of abscisic acid (ABA) signalling. 2. The oxidative signal is propagated along the embryonic axis to the hypocotyl area. 3. At the onset of radicle protrusion cells elongate in the hypocotyl region after cell wall loosening caused by ROS production and apoplastic ROS (aROS) generation in the cell wall. Superoxide dismutase (SOD), catalase (CAT) and glutathion reductase (GR) are activated to prevent excessive ROS accumulation. Germination can then proceeds.

.

by H 2 O

 2 2 accumulation and relay ROS signals [100], sometimes in connexion with hormone signaling [101]. H 2 O 2 and Ca 2+ signaling pathways are closely linked and can act in concert to regulate the effect of plant hormones, such as the one of ABA on stomatal closure in response to drought [1]. Many examples have also highlighted that the relationship between NO and H 2 O 2 can influence various plant developmental and processes and responses to biotic and abiotic factors [102,103]. A set of transcription factors relay the ROS message to transcriptome reprograming [104]. Members of MYB, DREB, ZAT, bHLH, WRKY, bZIP, and NAC families have been reported to be associated with ROS signalling in plants [1,105].

Figure 1 .

 1 Figure 1. Dynamics of ROS production during Arabidopsis seed germination. A-C, ROS detection in the embryonic axis of non-dormant seeds after 6 (A), 16 (B) and 24 h (C) of imbibition at 25°C in the darkness. D-F, fluorescence intensity along a line running from the radicle tip (rt) to the hypocotyl shootward region (h), as shown in A-C, in seeds imbibed for 6 (D), 16 (E) and 24 h (F). ROS production was detected by fluorescence using 5-(and-6)-chloromethyl-20,70-dichlorofluorescein diacetate (DCFH-DA) and visualized by

Figure 2 . 1 .Figure 1 .Figure 2 .

 2112 Figure 2. A spatiotemporal model explaining the signalling role of ROS in seed germination. 1. At the beginning of imbibition, environmental conditions are perceived by the imbibed seed at the meristematic region where a limited ROS production occurs through mitochondria electron transfer chain and NADPH oxidase activity. It induces the oxidation of negative regulators of germination, activation of gibberellin (GA) signalling and inactivation of abscisic acid (ABA) signalling. 2. The oxidative signal is propagated along the embryonic axis to the hypocotyl area. 3. At the onset of radicle protrusion cells elongate in the hypocotyl region after cell wall loosening caused by ROS production and apoplastic ROS (aROS) generation in the cell wall. Superoxide dismutase (SOD), catalase (CAT) and glutathion reductase (GR) are activated to prevent excessive ROS accumulation. Germination can then proceeds.

Table 2

 2 mRNA abundance in the translatome of dormant and non-dormant Arabidopsis seeds compared to the clusters from the ROS wheel analysis of ROS responses [108]. GUN, genome uncoupled mutants (known plastid retrograde signaling components); HL Late, 3 to 8 h of high light exposure; HL Early, 30 min to 2 h high light exposure; ROS cell culture, mitochondrial electron transport/ATP synthase inhibitor or H 2 O 2 treatment of cell cultures; ROS, direct application or indirect generation of ROS in plants; 1 O 2 , exposure to singlet oxygen; UVB Early, 15 min to 1 h of UV-B exposure, RBOHF, responses in rbohF mutant background; ROS Acclimation, redox mutants leading to long-term ROS stress. Translatome data are from Basbouss-Serhal et al. [109]. Dormant and non-dormant seeds were imbibed for 16 and 24 h at 25°C in the darkness.

	ROS wheel	mRNAs specifically found in the translatome of
	clusters	non-dormant seeds at	dormant seeds at
		16 h of	24 h of	16 h of	24 h of
		imbibition	imbibition	imbibition	imbibition
	I-GUN
	retrograde	22	2	4	4
	II -HL late	3	3	1	3
	III -HL early	36	14	79	61
	IV -ROS cell
	culture	1	0	7	5
	V -ROS	0	0	1	1
	VI -1 O 2 -UVB
	early	0	0	1	2
	VII -RBOHF	0	0	0	0
	VIII -ROS
	acclimation	1	1	1	0
	Total	63	20	94	76

Acknowledgments

The author thanks Rana Jurdark and Huifang Yang for her help with confocal imaging, Maharajah Ponnaiah for his help with the ROS wheel and George W. Bassel for critical reading of the manuscript

Competing Interests

The Author declares that there are no competing interests associated with the manuscript. Corbineau, F., Meimoun, P. and El-Maarouf-Bouteau, H. (2018) Integrating proteomics and enzymatic profiling to decipher seed metabolism affected by temperature in seed dormancy and germination. Plant Sci. Int. J. Exp. Plant Biol. 269, 118-125. Mesembryanthemum crystallinum [START_REF] Visscher | Dry heat exposure increases hydrogen peroxide levels and breaks physiological seed coat-imposed dormancy in Mesembryanthemum crystallinum (Aizoaceae) seeds[END_REF] Drought and salt stress negative Miscanthus [START_REF] Yang | Miscanthus NAC transcription factor MlNAC12 positively mediates abiotic stress tolerance in transgenic Arabidopsis[END_REF] Germination/ABA positive pea [START_REF] Barba-Espin | Role of thioproline on seed germination: Interaction ROS-ABA and effects on antioxidative metabolism[END_REF] Germination positive Pea [START_REF] Kranner | Extracellular production of reactive oxygen species during seed germination and early seedling growth in Pisum sativum[END_REF] High temperature, drought stress negative rice [START_REF] Liu | High temperature and drought stress cause abscisic acid and reactive oxygen species accumulation and suppress seed germination growth in rice[END_REF] Low phytic acid seed vigour positive rice [START_REF] Zhou | Suppression of ROS generation mediated by higher InsP(3) level is critical for the delay of seed germination in lpa rice[END_REF] Dormancy alleviation (after-ripening) positive rice [START_REF] Zhang | Carbonylated protein changes between active germinated embryos and quiescent embryos give insights into rice seed germination regulation[END_REF] Germination / NADPH oxidase positive rice [START_REF] Li | Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination[END_REF] Osmotic and salt stress negative rice [START_REF] Chen | Specific roles of tocopherols and tocotrienols in seed longevity and germination tolerance to abiotic stress in transgenic rice[END_REF] Germination /ABA/GA positive rice [START_REF] Ye | Antagonism between abscisic acid and gibberellins is partially mediated by ascorbic acid during seed germination in rice[END_REF] Germination


Marco Marazzi

Antonio Francés-Monerris

Maxime Mourer

Andreea Pasc

Antonio Monari

Trans-to-cis photoisomerization of cyclocurcumin in different environments rationalized by computational photochemistry

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The extracts of Curcuma longa, also known as turmeric, [START_REF] Sasikumar | Handbook of Herbs and Spices: Second Edition[END_REF][START_REF] Cas | Dietary Curcumin: Correlation between Bioavailability and Health Potential[END_REF] have been widely used for centuries both as spices and food colouring agents. [START_REF] Priyadarsini | The Chemistry of Curcumin: From Extraction to Therapeutic Agent[END_REF] They are characterized by a rather intense and typical yellow colour due to the presence of conjugated polyphenol backbone characteristic of curcuminoid. While the most abundant component is the curcumin and their closely related derivatives, in the nineties an additional component known as cyclocurcumin was isolated and its structure was reported. In addition to its use in food industry, turmeric derivatives, and curcumin in particular, have started attracting considerable interest due to their biological and pharmaceutical properties. [START_REF] Kuptniratsaikul | Efficacy and Safety of Curcuma Domestica Extracts Compared with Ibuprofen in Patients with Knee Osteoarthritis: A Please do not adjust margins Multicenter Study[END_REF][START_REF] Mazzolani | Oral Administration of a Curcumin-Phospholipid Delivery System for the Treatment of Central Serous Chorioretinopathy: A 12-Month Follow-up Study[END_REF][START_REF] Aggarwal | Potential Therapeutic Effects of Curcumin, the Anti-Inflammatory Agent, against Neurodegenerative, Cardiovascular, Pulmonary, Metabolic, Autoimmune and Neoplastic Diseases[END_REF][START_REF] Reddy | Curcumin for Malaria Therapy[END_REF][START_REF] Wright | Bioactivity of Turmeric-Derived Curcuminoids and Related Metabolites in Breast Cancer[END_REF][START_REF] Vera-Ramirez | Curcumin and Liver Disease[END_REF][START_REF] Hewlings | Curcumin: A Review of Its' Effects on Human Health[END_REF] Indeed, the antioxidant, antibacterial and antiviral properties of curcumin have been unequivocally demonstrated. [START_REF] Hewlings | Curcumin: A Review of Its' Effects on Human Health[END_REF] Moreover, its absorption in the visible range makes turmeric extracts, including curcumin, a most valuable candidate for its use in photodynamic therapy (PDT), [START_REF] Ormond | Dye Sensitizers for Photodynamic Therapy[END_REF] or for light-assisted food decontamination, [START_REF] Penha | Photodynamic Inactivation of Foodborne and Food Spoilage Bacteria by Curcumin[END_REF][START_REF] Brovko | Antimicrobial Light-Activated Materials: Towards Application for Food and Environmental Safety[END_REF] despite its rather limited solubility in aqueous environment, that usually requires the development of appropriate drug delivering strategies. [START_REF] Mangolim | Curcumin-β-Cyclodextrin Inclusion Complex: Stability, Solubility, Characterisation by FT-IR, FT-Raman, X-Ray Diffraction and Photoacoustic Spectroscopy, and Food Application[END_REF][START_REF] Cui | Enhancement of Oral Absorption of Curcumin by Self-Microemulsifying Drug Delivery Systems[END_REF][START_REF] Sun | A Novel Nanoparticle Drug Delivery System: The Anti-Inflammatory Activity of Curcumin Is Enhanced When Encapsulated in Exosomes[END_REF][START_REF] Anitha | Efficient Water Soluble O-Carboxymethyl Chitosan Nanocarrier for the Delivery of Curcumin to Cancer Cells[END_REF][START_REF] Bansal | Advanced Drug Delivery Systems of Curcumin for Cancer Chemoprevention[END_REF][START_REF] Gupta | Therapeutic Roles of Curcumin: Lessons Learned from Clinical Trials[END_REF] Curcumin-primed exosomes have also been used in vivo to treat neuro-degenerative disorder in mice showing extremely promising potentiality in alleviating the symptoms of Alzheimer's disease. [START_REF] Wang | Curcumin-Primed Exosomes Potently Ameliorate Cognitive Function in AD Mice by Inhibiting Hyperphosphorylation of the Tau Protein through the AKT/GSK-3β Pathway[END_REF] The photophysics of curcumin and its complex modulation due to the interplay between the packing in liposomes, and the modulation of keto-enol tautomerism has also been recently addressed by a combination of spectroscopic and computational techniques. [START_REF] Girardon | Triggering Tautomerization of Curcumin by Confinement into Liposomes[END_REF] In contrast, the biological properties of cyclocurcumin are much less characterized than the ones of its parent compound, and its use is much more limited. [START_REF] Kim | an Antivasoconstrictive Constituent of Curcuma Longa (Turmeric)[END_REF][START_REF] Fu | Cyclocurcumin, a Curcumin Derivative, Exhibitsimmune-Modulating Ability and Is a Potential Compound for the Treatment of Rheumatoid Arthritisas Predicted by the MM-PBSA Method[END_REF] Indeed, a more limited anticancer activity compared to curcumin has been recognized for cyclocurcumin against the proliferation of brain cancer cell lines. [START_REF] Chakraborty | Inhibitory Effects of Curcumin and Cyclocurcumin in 1-Methyl-4-Phenylpyridinium (MPP+) Induced Neurotoxicity in Differentiated PC12[END_REF][START_REF] Ravindran | Curcumin and Cancer Cells: How Many Ways Can Curry Kill Tumor Cells Selectively?[END_REF] Nevertheless, the antiproliferative activity of curcuminoids increases sensibly when they are applied in combination, suggesting a synergic effect. Cyclocurcumin photoisomerization of the double bond around the dihedral angle q is envisaged to be responsible for the process.

As it can be inferred from Figure 1, cyclocurcumin is characterized by the presence of a carbon-carbon double bond that gives rise to the coexistence of cis and trans isomers. As it Please do not adjust margins Please do not adjust margins is evident from sterical considerations, the trans isomer is the thermodynamically favoured one and it is indeed the one present in the natural extract. Compounds presenting isomerizable double bonds within rather extended pconjugated backbones, such as carotenoids and retinal, [START_REF] Hubbard | Cis-Trans Isomers of Vitamin A and Retinene in the Rhodopsin System[END_REF][START_REF] Wald | Molecular Basis of Visual Excitation[END_REF][START_REF] Altoè | Aborted Double Bicycle-Pedal Isomerization with Hydrogen Bond Breaking Is the Primary Event of Bacteriorhodopsin Proton Pumping[END_REF][START_REF] Briand | Coherent Ultrafast Torsional Motion and Isomerization of a Biomimetic Dipolar Photoswitch[END_REF][START_REF] González-Luque | Computational Evidence in Favor of a Two-State, Two-Mode Model of the Retinal Chromophore Photoisomerization[END_REF][START_REF] Tomasello | Electrostatic Control of the Photoisomerization Efficiency and Optical Properties in Visual Pigments: On the Role of Counterion Quenching[END_REF][START_REF] Aman | Effects of Heating and Illumination on Trans-Cis Isomerization and Degradation of β-Carotene and Lutein in Isolated Spinach Chloroplasts[END_REF] or azobenzene [START_REF] Aleotti | Multidimensional Potential Energy Surfaces Resolved at the RASPT2 Level for Accurate Photoinduced Isomerization Dynamics of Azobenzene[END_REF][START_REF] Tiberio | How Does the Trans-Cis Photoisomerization of Azobenzene Take Place in Organic Solvents?[END_REF][START_REF] Bandara | Photoisomerization in Different Classes of Azobenzene[END_REF] and stilbene [START_REF] Waldeck | Photoisomerization Dynamics of Stilbenes[END_REF][START_REF] Bylina | Photo-Isomerization and the Triplet State of Stilbene[END_REF][START_REF] Tomasello | Tracking the Stilbene Photoisomerization in the S1 State Using RASSCF[END_REF] are regarded as paradigmatic molecular photoswitches, [START_REF] García-Iriepa | Z Photochemical Switches: Syntheses, Properties and Applications[END_REF][START_REF] Marazzi | Retinal Inspired Photoswitches[END_REF] i.e. compounds able to undergo photoisomerization after the absorption of visible light. The study of natural photoswitches has been extremely active and has allowed to important breakthroughs in the last years, also allowing the engineering of bio-inspired synthetic photoswitches. [START_REF] Garcia-Iriepa | A Biomimetic Molecular Switch at Work: Coupling Photoisomerization Dynamics to Peptide Structural Rearrangement[END_REF][START_REF] Sampedro | Design and Photochemical Characterization of a Biomimetic Light-Driven Z/E Switcher[END_REF] In this respect, cyclocurcumin presents an almost optimal molecular structure, with the presence of only one isomerizable double bond, despite a non-negligible overlap between the absorption spectra of the two isomers. The presence of a single isomerizable bond is highly desirable, since it allows a fine control of the photoswitching activity between two defined states (i.e. trans and cis isomers, avoiding the complexity due to a higher number of states). However, and despite its attractiveness, its photophysical properties have been only marginally addressed in the literature. In particular, Adhikary et al. [START_REF] Adhikary | Photoinduced Trans-to-Cis Isomerization of Cyclocurcumin[END_REF] have reported the influence of the molecular environment, and specifically of the polarity of the solvent, on the fluorescence quantum yield of cyclocurcumin. By low temperature fluorescence decay analysis the authors have also concluded that cyclocurcumin is most likely performing transto-cis photoisomerization in polar solvents, only pointing to a strong dependence on the polarity and the viscosity of the medium. [START_REF] Adhikary | Photoinduced Trans-to-Cis Isomerization of Cyclocurcumin[END_REF] However, the fine molecular and electronic factors hiding behind the environmental dependence of the photoisomerization efficiency, are not rationalized, yet. In particular, no proper characterization of the potential energy surfaces (PES) of the different excited states involved in the process, and their modulation by the molecular surroundings has been provided.

Figure 2) TD-DFT calculated absorption spectrum of cyclocurcumin in gas phase (A) and in different solvents (B). A comparison between B3LYP and CAM-B3LYP functionals is shown for the gas phase spectrum, while the CAM-B3LYP functional was used to consider the solvent effect

In this contribution we aim at filling this gap by the use of highlevel computational photochemistry techniques to finely characterize the isomerization process in terms of the nature of the involved excited states, the topology of the PES, and the energy barriers needed to be overcome to finally reach the nonadiabatic crossing regions driving the photochemical process. In this respect, our results will be important to pave the way to the possible exploitation of cyclocurcumin as PDT agents or in photo-triggered drug-delivery strategies. Indeed, cyclocurcumin could destabilize biological lipid membranes through its photoinduced isomerization leading to their apoptosis. The same phenomenon could also be used to force the controlled release of active drugs incorporated in liposomes. However, such a strategy could be reasonably pursued only after a preliminary comprehension of the photochemical pathways of cyclocurcumin, as well as of the influence of complex inhomogeneous environments on such phenomena.

Results and discussion

The experimental absorption spectrum of cyclocurcumin, most notably reported by Adhikary et al, [START_REF] Adhikary | Photoinduced Trans-to-Cis Isomerization of Cyclocurcumin[END_REF] is characterized by an intense maximum at around 375 nm that is slightly red-shifted up to 380-385 nm upon increasing the polarity of the medium. The calculated absorption spectrum, taking into account the vibrational and dynamical effects thanks to the sampling of the ground state conformational space via a Wigner distribution, is reported in Figure 2, while the absorption spectrum for the cis isomer is reported in Supplementary Information.

The spectrum in gas phase for the trans isomer calculated using both B3LYP and CAM-B3LYP functionals is reported. Even if the former provides a globally red-shifted spectrum that is closer to the experimental absorption wavelengths (360 nm), it is not able to recover the correct energy ordering of the excited states leading to an accumulation of partial charge-transfer states in the red tail of the spectrum. For this reason, in the following only the results at the CAM-B3LYP level of theory will be considered. The spectrum of the cis isomer shows a globally decreased oscillator strength, even if a non-negligible overlap with the absorption band of the trans isomer is still observed. The CAM-B3LYP simulated absorption spectrum in the gas phase is characterized by an intense maximum at around 350 nm followed by an evident tail in the red region extending up to 400-450 nm. Such a band shape is indeed characteristic of the presence of a bright 1 (p,p*) state (S2) and a darker and lower in energy [START_REF] Sasikumar | Handbook of Herbs and Spices: Second Edition[END_REF] (n,p*) state (S1). The nature of the states is also confirmed by the analysis of the Natural Transition Orbitals (NTOs) describing the first two excited states as reported in Figure 3, and by the analysis of the oscillator strengths that are of 0.06 and 1.16 for S1 and S2, respectively. Please do not adjust margins

Please do not adjust margins

Figure 3) NTOs describing the first two excited states obtained from TD-DFT calculation in gas phase and using the CAM-B3LYP functional.

The calculation of the topological index fS, which indicates the charge transfer character of the states, also gives a coherent picture with a value of 0.5 for S1, pointing to a moderate charge redistribution, and 0.9, hence an almost pure locally excited state, for S2. As reported in Figure 2B, and once again coherently with the experimental results, polar solvents induce a slight redshift of the absorption maximum, however without changing the main characteristics of the band shape. When compared with experimental results [START_REF] Adhikary | Photoinduced Trans-to-Cis Isomerization of Cyclocurcumin[END_REF] , the simulated spectrum appears in good agreement in terms of the maximum absorption wavelengths and of the band shape, even though the CAM-B3LYP functional provides an absorption maximum that is slightly blue-shifted of about 10 nm (0.2 eV) compared to the experimental results. On the other hand, the experimental solvatochromism is less well reproduced, this fact being most probably due to the use of a continuum model for the solvent and hence neglecting some micro-solvation effects that may become more important for polar and protic media. The calculated values for the fluorescence spectra also compare reasonably well with experimental results. [START_REF] Sampedro | Design and Photochemical Characterization of a Biomimetic Light-Driven Z/E Switcher[END_REF] Indeed, while in gas phase or apolar solvents we obtain an experimental and calculated emission maximum at 420 and 397 nm, respectively, in the case of ethanol, the experimental and calculated maxima are shifted to 500 and 466 nm, respectively. The larger discrepancy with the experimental values is also due to the fact that the computational maxima are obtained as vertical transitions from the S1 optimized geometry, hence neglecting vibrational and dynamic effects, usually leading to a red shift. [START_REF] Gattuso | Photophysics of Chlorin E6: From One-and Two-Photon Absorption to Fluorescence and Phosphorescence[END_REF][START_REF] Moron | Far Red Fluorescent Proteins: Where Is the Limit of the Acylimine Chromophore?[END_REF] As reported in Table 1, the order and the nature of the states provided by CAM-B3LYP is also confirmed by using multiconfigurational complete active space methods. The nature of the states is, once more, confirmed by the analysis of the weight of the molecular orbitals in the CAS expansion (see Supplementary Information). The analysis of the PES for the two low-lying excited states leads to the obtention of two minima that are quite close to the Franck-Condon geometry.

In particular, the 1 (p,p*) minimum is almost totally planar presenting a dihedral angle around the isomerizable doublebond (q) of 179°, while the optimization of the 1 (n,p*) state leads to a slightly more twisted conformation with an equilibrium value of q = 176°. The changes in the q angle are coupled with the increases of the bond length of the isomerizable double bond -of 0.05 and 0.07 Å for 1 (n,p*) and 1 (p,p*) minima, respectively. This fact is coherent with the decrease of the double-bond character and the induction of a noticeable bond length alternation. This geometrical deformation already points to the possible triggering of a photoisomerization process, however the presence of a minimum in the PES may also preclude the existence of ultrafast, i.e. sub ps, processes. Interestingly, an accessible S2/S1 conical intersection (CI) is also noticed when irradiating to S2, with the diabatic 1 (p,p*) state becoming the first excited state at the corresponding optimized geometry. As a consequence, the participation of both states to the photoisomerization process should be taken into account.

To follow the photoisomerization process in terms of the exploration of the relevant region of the PES of the two lowestenergy excited states, we firstly calculated the relaxed scan along the torsion of the dihedral angle q (see Figure 1) starting from the respective excited state minima, selecting the Please do not adjust margins Please do not adjust margins multistate MS-CASPT2 method in gas phase. Note that, the choice to consider only the value of the dihedral angle to examine the PESs is due to the fact that it is the most relevant (i.e. representative) coordinate in the description of the transcis isomerization. Indeed, as in other well-studied photoswitches (e.g. retinal), [START_REF] Marazzi | Retinal Inspired Photoswitches[END_REF][START_REF] Guo | Different Hydrogen Bonding Environments of the Retinal Protonated Schiff Base Control the Photoisomerization in Channelrhodopsin-2[END_REF][START_REF] Hontani | Reaction Dynamics of the Chimeric Channelrhodopsin[END_REF] bond length alternation of single and double bonds is the initial driving force taking place after photon absorption, leading to torsion of the formal double bond implied in the photoisomerization. To discriminate between the participation of the two excited states in the photoprocess, we reported the pathways calculated following the relaxation along the 1 (n,p*), displayed in Figure 4A, and the 1 (p,p*) state, shown in Figure 4B. In both profiles, one can notice the presence of crossing points between the S0 and S1 PES, i.e. structures that mediate the excited-state decay via photoisomerization. However, the S1/S0 CI is reached earlier, i.e. at a lower q twisting, for the 1 (p,p*) profile, around 115°, whereas the 1 (n,p*) state requires a more pronounced torsion of about 90° (Figure 4B). This fact could lead to a decrease in the photoisomerization quantum yield for the 1 (p,p*) profile, due to the occurrence of a considerable number of frustrated hops that would revert to the original trans isomer instead than completing the photoisomerization. On the other hand, the profile to reach the S1/S0 CI from the 1 (n,p*) profile is much steeper than the corresponding one calculated for the 1 (p,p*) state, which has a larger excitation energy according to the vertical absorption energies summarised in Table 1. Indeed, while in the former a barrier of about 0.7 eV needs to be overcome to reach the non-adiabatic point, in the latter the energy penalty is reduced to ca 0.4 eV. Hence, while the photoisomerization from the 1 (n,p*) state is much less competitive based on energetic considerations, and the system in this state will most likely be trapped around the S1 minimum and fluoresce, the photoisomerization pathway is more affordable along the 1 (p,p*) state, even if the 0.4 eV energy barrier found for this profile will globally delay the photoprocess to the picosecond scale.

Figure 5) Energy profiles along the torsion of the dihedral angle q leading to the photoisomerization, calculated in gas phase at TD-DFT level (using the CAM-B3LYP functional) and following the 1 (n,p*) (A) and the 1 (p,p*) excited state (B).

Moreover, the moderate distortion of the S1/S0 CI geometry should also decrease the efficiency of the photoprocess. The profile reported in Figure 4B is reminiscent of the peculiar properties of the retinal chromophore when forming the active site of channelrhodopsin [START_REF] Hontani | Reaction Dynamics of the Chimeric Channelrhodopsin[END_REF][START_REF] Guo | Active Site Structure and Absorption Spectrum of Channelrhodopsin-2 Wild-Type and C128T Mutant[END_REF][START_REF] Welke | QM/MM Simulations of Vibrational Spectra of Bacteriorhodopsin and Channelrhodopsin-2[END_REF] and should share the same photochemical characteristics, although a lower excited state barrier in channelrhodopsin can be associated with a faster photoisomerization process (S1 lifetime of ca. 400 fs). Note also that the crossing between the 1 (n,p*) and the 1 (p,p*) states takes place at coordinates extremely close to the Franck-Condon region, where the two states are characterized by an extended region of quasi-degeneracy in which the PES evolve almost in parallel. Similar profiles are obtained with the TD-DFT method, as reported in Figure 5. Conversely, the extended multi state XMS-CASPT2 method (see Supplementary Information), while giving the same picture for the 1 (n,p*) profiles, provides a crossing between the two excited states shifted to smaller q values in the case of the 1 (p,p*) pathway.

After having assessed the gas phase photochemistry of cyclocurcumin, the important environmental effects observed experimentally can now be addressed and be related to the influence that the electrostatic field generated by the different solvents on the topology of the PES. As a matter of fact, this influence may lead to state inversion and hence to the tuning of the different accessible photochemical pathways.

In Figure 6 we report the most probable pathway leading to the S1/S0 CI structures that trigger the photoisomerization, calculated in non-polar (cyclohexane), polar (acetonitrile), and protic (ethanol and water) solvents. As expected, the behaviour of cyclocurcumin is strongly modified depending on the molecular surroundings. In the non-polar medium at Franck-Condon region, both 1 (n,p*) and 1 (p,p*) states could be initially populated, leading in any case to a rather steep PES leading first to a 1 (n,p*)/ 1 (p,p*) crossing (at around q = 140°) and subsequently to the S1/S0 CI allowing photoisomerization. In contrast, for polar and protic solvents, the optically brighter 1 (p,p*) state is stabilized in energy, becoming the S1 state at Franck-Condon region, largely increasing the probability to be initially populated when irradiating the compound. Moreover, the Franck-Condon and the S1/S0 CI structures are connected through globally barrierless or almost barrierless PESs, thus leading to photoisomerization. As a consequence, the 1 (n,p*) state is destabilized in polar solvents precluding any 1 (n,p*)/ 1 (p,p*) crossing.

Figure 6) Energy profiles along the torsion of the dihedral angle q calculated at TD-DFT level in presenc e of different solvents

Cyclohexane Acetonitrile Ethanol Water

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This means that, in polar and protic solvents, cyclocurcumin photophysics can be considered as a 2-(electronic) states system. In stark contrast, in non-polar environments cyclocurcumin is a 3-states system, mostly trapped in the region of the 1 (n,p*) minimum and hence giving rise to fluorescence. On the contrary, polar media favours photoisomerization over luminescence, and the fluorescence quantum yield is significantly decreased. This picture is in perfect agreement with the experimental results of Adhikary et al., [START_REF] Adhikary | Photoinduced Trans-to-Cis Isomerization of Cyclocurcumin[END_REF] who have clearly shown fluorescence quenching when increasing the solvent polarity, whereas the radiative decay is active in cyclohexane. Hence, it is clear that polar solvents will activate non-radiative decay pathways leading to trans-to-cis isomerization, consistently diminishing the lifetime of the excited states and preventing luminescence. In addition, since the isomerization is proceeding through a rather pronounced spatial movement involving large molecular fragments, the viscosity of the medium is supposed to play a fundamental role as well.

Our results offer a clear electronic-based picture of the characteristic behaviour of cyclocurcumin. As an important consequence, the possible modulation of the photochemical properties by the environment could qualify cyclocurcumin as a bifunctional photoswitch and could potentially be exploited for therapeutic purposes in light-assisted chemotherapy. Indeed, in the complex and inhomogeneous environment, offered for instance by lipid bilayers, it is expected that the delicate equilibrium between luminescence and photoisomerization will become even more complex and could also depend on the position of cyclocurcumin with respect to the membrane axis. However, the coexistence of isomerization and fluorescence can be exploited to combine diagnosis and treatment, as in a theranostic-like approach. Furthermore, preliminary TD-DFT calculations performed at the Franck-Condon geometry have shown that cyclocurcumin has a non-negligible two-photon absorption (TPA), allowing to reach the infrared region. Indeed, the calculated cross section for TPA, in gas phase, amounts to 14 GM, a value that is relatively high for simple organic molecules, offering attractive therapeutic possibilities for this compound. Even though the total elucidation of the non-linear optical properties of cyclocurcumin and their dependence on the environment is well beyond the scope of the present contribution, the calculated values of the TPA cross-section and related wavelengths, together with its inherent photochemistry, definitively suggest the possible use of cyclocurcumin as a TPA active drug that may be operative in the therapeutic window, also exploiting a much higher spatial resolution thanks to the non-linear nature of TPA.

Conclusions

By using molecular modelling techniques, and in particular by a proper exploration of the nature and topology of the excited states of cyclocurcumin, including the exploration of the PES along relevant coordinates, we have clearly demonstrated its capability to act as a photoisomerizable switch. In particular, we have shown the relevant role played by the two lowest excited states, 1 (n,p*) and 1 (p,p*), and how the photoisomerization will be favoured while proceeding on the 1 (p,p*) PES. In turn, we have also shown how the photoresponse is strongly modified by the molecular environment: polar solvents favour the population of the 1 (p,p*) state, hence leading to a flatter PES giving access to the S1/S0 CI structures that trigger photoisomerization, while non-polar solvents stabilize the 1 (n,p*) state hampering the access to the crossing region with the ground state. As a consequence, while in polar solvents cyclocurcumin is expected to photoisomerize, albeit not at an ultrafast time scale, in non-polar environments the fluorescence will be dominant. This bifunctional nature of cyclocurcumin could lead to fascinating applications in lightassisted chemotherapy and in theranostics, also thanks to the previewed relatively high TPA cross section that could allow its activation via infrared light, hence covering the biological therapeutic window. All in all, our results clearly point to the possible use of cyclocurcumin for biotechnological or, in the longer time scale, medical purposes in particular exploiting its photoisomerization. Cyclocurcumin can offer an alternative photochemical pathway compared to other turmeric extracts, such as curcumin, whose light-activated mechanism of action is dominated by intersystem crossing and hence the activation of molecular oxygen through Type II photosensitization. Hence, cyclocurcumin could be efficiently used in conditions of hypoxic solid tumours that can be resistant to conventional PDT. However, the present results also offer some important suggestions on possible rational drug design strategies that should be pursued to maximize cyclocurcumin efficiency, also going above the simple, yet crucial, increase of its bioavailability. In particular, one can imagine to selectively stabilize the pp* state over the np* to increase the photoisomerization quantum yield. As an alternative application, the environmental dependence of photoisomerization and fluorescence quantum yield with the polarity of the medium points to the potential application of cyclocurcumin derivates as chemosensors, detecting in real time polarity changes in micro-environments. [START_REF] Yang | Macro-/Micro-Environment-Sensitive Chemosensing and Biological Imaging[END_REF] In the future, we plan to carefully investigate the properties of cyclocurcumin and its derivatives for their inclusion in lipid bilayers, mimicking liposomes or biological membranes, using both experimental time-resolved spectroscopy techniques and hybrid quantum mechanics/molecular mechanics simulations to assess the effects of inhomogeneous environments on its photochemical properties. In the same way, the influence of the dynamical and vibrational degrees of freedom and of the complex surroundings on TPA will also be pursued, including the eventual sensitization of cyclocurcumin with specific TPA antennas to increase the relevance of this feature.

Computational Methodology

Transand cis-cyclocurcumin ground state minimum structures were located by Density Functional Theory (DFT), by applying the B3LYP functional. 51 The absorption spectrum of both isomers was calculated by using the Time Dependent flavour of DFT (TD-DFT), using B3LYP 51 and CAM-B3LYP [START_REF] Yanai | A New Hybrid Exchange-Correlation Functional Using the Coulomb-Attenuating Method (CAM-B3LYP)[END_REF] functionals. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins More in detail, the ground state phase space was sampled around the Franck-Condon structure via a Wigner distribution, using the NewtonX code [START_REF] Barbatti | Newton-X: A Surface-Hopping Program for Nonadiabatic Molecular Dynamics[END_REF] to generate 100 structures. Excited states were computed for each structure through single point calculations, finally convoluting all Gaussian functions resulting from all transition energies and oscillator strengths (full width at half maximum = 0.2 eV). This approach to simulate the absorption spectrum of organic chromophores was proven to be adequate by some of the authors. [START_REF] Gattuso | Photophysics of Chlorin E6: From One-and Two-Photon Absorption to Fluorescence and Phosphorescence[END_REF][START_REF] Gattuso | Two-Photon-Absorption DNA Sensitization via Solvated Electrons Production: Unraveling the Photochemical Pathways by Molecular Modeling and Simulation[END_REF][START_REF] Assfeld | Steady-State Linear and Non-Linear Optical Spectroscopy of Organic Chromophores and Bio-Macromolecules[END_REF][START_REF] Turan | Assessing One-and Two-Photon Optical Properties of Boron Containing Arenes[END_REF][START_REF] Koca | Exploring the Photophysics of Polyfluorinated Phthalocyanine Derivatives as Potential Theranostic Agents[END_REF] To properly select a convenient active space for multi-configurational methods, the absorption spectrum was calculated also for the B3LYP Franck-Condon structure at MS-CASPT2//SA(4)-CASSCF level, [START_REF] Andersson | Second-Order Perturbation Theory with a CASSCF Reference Function[END_REF][START_REF] Andersson | Second-Order Perturbation Theory with a Complete Active Space Self-Consistent Field Reference Function[END_REF] i.e. a first Complete Active Space Self-Consistent Field calculation was performed including 4 averaged states (from S0 to S3), followed by a multistate calculation applying 2 nd order perturbation theory to the CASSCF wavefunction. As shown in Figure S3 of the Supplementary Information, three active states were tested, in all cases including a n orbital and several π and π* orbitals: 10 electrons in 9 orbitals (10,9), 12 electrons in 11 orbitals (12,11), and 14 electrons in 13 orbitals (14,13). All active spaces predict a bright S0→S2 vertical transition, whereas the S0→S1 is notably dark. This finding is coherent with the fact that in gas phase the S0→S1 transition is of a 1 (n,π*) nature, while S0→S2 is of 1 (π,π*) character.

Hence, as a compromise between accuracy and required computational cost, the photochemistry of cyclocurcumin was studied with the (10,9) active space. In particular, the S1 (np*) and S2 (pp*) states identified at the Franck-Condon region were optimized with the CASSCF method, reaching the respective minima whose diabatic nature was confirmed by analysing the respective molecular orbitals. Starting from these structures, we performed relaxed scans along the dihedral angle q optimizing both S1 and S2 states. In addition to multiconfigurational method TD-DFT calculations were also performed.

On top of all CASSCF optimized structures, single point energy calculations were performed at different level of theory in order to compute the necessary dynamic electron correlation: MS-CASPT2, extended (X)MS-CASPT2, [START_REF] Shiozaki | Extended Please do not adjust margins Multi-State Complete Active Space Second-Order Perturbation Theory: Energy and Nuclear Gradients[END_REF] and CAM-B3LYP (with and without spinflip), in gas phase. Considering the correct description given by CAM-B3LYP, solvent effects were included at this level of theory: cyclohexane, acetonitrile, ethanol and water were considered by applying the equilibrium state-specific Polarizable Continuum Model (PCM) within the integral equation formalism variant (IEFPCM). [START_REF] Mennucci | Polarizable Continuum Model[END_REF][START_REF] Tomasi | Quantum Mechanical Continuum Solvation Models[END_REF] Although TD-DFT is problematic in correctly describing S1/S0 CIs due to the lack of multiconfigurational character for the ground state, [START_REF] Levine | Conical Intersections and Double Excitations in Time-Dependent Density Functional Theory[END_REF] it has to be noted that our TD-DFT results are obtained on top of CASSCF optimized geometries (i.e. TD-DFT//CASSCF approach), and that the gas phase PES obtained at TD-DFT and CASPT2 level agrees considerably, pointing to the robustness of our approach. To further confirm this point, we have also repeated the calculation of the gas phase PES using a spin-flip TD-DFT [START_REF] Levine | Conical Intersections and Double Excitations in Time-Dependent Density Functional Theory[END_REF][START_REF] Gozem | Shape of Multireference, Equationof-Motion Coupled-Cluster, and Density Functional Theory Potential Energy Surfaces at a Conical Intersection[END_REF][START_REF] Zhang | Analytic Derivative Couplings for Spin-Flip Configuration Interaction Singles and Spin-Flip Time-Dependent Density Functional Theory[END_REF][START_REF] Minezawa | Optimizing Conical Intersections by Spin-Flip Density Functional Theory: Application to Ethylene[END_REF] //CASSCF approach (Supplementary Information) that also provides a coherent picture with both multiconfigurational and TD-DFT results.

For all multi-configurational calculations, the 6-31G* basis set was adopted, while the 6-311+G* basis set was used for DFT and TD-DFT calculations. Spin-flip TD-DFT calculations were performed with the TZP basis set.

In the case of MS-and XMS-CASPT2 calculations, no IPEA was applied, while an imaginary shift of 0.2 a.u. was considered to avoid intruder states. Both MS-and XMS-CASPT2 methods were applied, since the latter is known to better perform in regions of energy degeneracy, [START_REF] Shiozaki | Extended Please do not adjust margins Multi-State Complete Active Space Second-Order Perturbation Theory: Energy and Nuclear Gradients[END_REF] while the former is more suitable to treat electronic states far in energy. Hence, the use of both methods was considered necessary to give an average correct description.

All CASSCF and CASPT2 calculations were run with the OpenMolcas suite of programs, [START_REF] Aquilante | Molcas 8: New Capabilities for Multiconfigurational Quantum Chemical Calculations across the Periodic Table[END_REF][START_REF] Fdez | OpenMolcas: From Source Code to Insight[END_REF] while DFT and TD-DFT calculations were performed with the Gaussian09 software. [START_REF] Frisch | Gaussian09 Revision D.01[END_REF] TPA cross-section have been determined at TD-DFT level using the Dalton package 70,71 and a protocol already successfully used for different organic dyes. [START_REF] Gattuso | Two-Photon-Absorption DNA Sensitization via Solvated Electrons Production: Unraveling the Photochemical Pathways by Molecular Modeling and Simulation[END_REF][START_REF] Turan | Assessing One-and Two-Photon Optical Properties of Boron Containing Arenes[END_REF] Spin-flip calculations were performed with ADF2018 modelling suite. [START_REF] Te Velde | Chemistry with ADF[END_REF] The charge transfer character of the excited states was evaluated at TD-DFT level by applying the topological index φS, [START_REF] Etienne | Toward a Quantitative Assessment of Electronic Transitions" Charge-Transfer Character[END_REF][START_REF] Etienne | New Insight into the Topology of Excited States through Detachment/Attachment Density Matrices-Based Centroids of Charge[END_REF][START_REF] Pastore | Unveiling the Nature of Post-Linear Response Z-Vector Method for Time-Dependent Density Functional Theory[END_REF] which tends to 1 (0) for locally (charge transfer) excited states, as it is an estimation of the overlap between occupied and virtual Natural Transition Orbitals (NTOs) [START_REF] Martin | Natural Transition Orbitals[END_REF] involved in the electronic transition. This journal is © The Royal Society of Chemistry 20xx

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Figure 1 )

 1 Figure 1) Molecular structure of trans-(A) and cis-(B) cyclocurcumin isomers.Cyclocurcumin photoisomerization of the double bond around the dihedral angle q is envisaged to be responsible for the process.

  a. Department of analytical chemistry, physical chemistry and chemical engineering, Universidad de Alcalá, Ctra. Madrid-Barcelona, Km 33,600, E-28805, Alcalá de Henares (Madrid), Spain. b. Chemical Research Institute "Andrés M. del Rio" (IQAR), Universidad de Alcalá, Ctra. Madrid-Barcelona, Km 33,600, E-28805, Alcalá de Henares (Madrid), Spain. c. Université de Lorraine and CNRS, LPCT UMR-7019, F-5400 Nancy, France. d. Departement de Quimica Fisica, Universitat de Valencia, 46100 Burjassot, Spain. e. Université de Lorraine and CNRS, L2CM UMR 7053, F-54000 Nancy, France. †Electronic Supplementary Information (ESI) available: multistate (MS)-CASPT2 benchmark with different active spaces; extended multistate (XMS)-CASPT2 energy pathways; simulated and experimental absorption spectrum of cis-cyclocurcumin; Cartesian coordinates (in Ångström) and 3D structures of the most relevant geometries along the photoreaction; spin-flip TD-DFT potential energy surfaces. See DOI: 10.1039/x0xx00000x 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Figure 4 )

 4 Figure4) S0→S1 (A) and S0→S2 (B) vertical transitions, followed by optimization of the populated 1 (n,p*) (A) and 1 (p,p*) (B) excited states. From the respective minima, a relaxed scan is performed along the torsion of the dihedral angle q, leading to the photoisomerization, as calculated in gas phase at MS-CASPT2 level.

  This journal is © The Royal Society of Chemistry 20xx

Table 1 )

 1 Relative energies of the ground and the two lowest-lying excited states of cyclocurcumin in gas-phase, calculated at different levels of theory and at different critical points on the PES.

	Level of theory Electronic	Franck-	1 (n,p*)	1 (p,p*)
		state	Condon	minimum	minimum
			geometry
	CASSCF	S0	0,00	1.36	0.97
		S1	4.17	2.51	3.60
		S2	5.40	4.74	4.40
	Multistate	S0	0.00	1.39	0.79
	(MS)-CASPT2	S1	3.51	3.15	3.29
		S2	4.04	4.58	3.52
	Extended	S0	0.00	1.44	0.38
	multistate	S1	3.69	3.39	3.65
	(XMS)-CASPT2	S2	4.49	4.96	4.28
	TD-DFT	S0	0.00	0.38	0.30
	(CAM-B3LYP)	S1	3.81	3.53	3.78
		S2	4.02	4.15	3.91

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Acknowledgements

Support from the Université de Lorraine and the French CNRS is gratefully acknowledged. DFT and TD-DFT calculations were performed on the LPCT local computing cluster and on the computing centre of the Université de Lorraine (Explor) in the framework of the project "Dancing Under the Light". CASSCF and CASPT2 calculations were performed on the local computing cluster of the Physical Chemistry Unit of the Universidad de Alcalá. A.F.-M. is grateful to the Generalitat Valenciana and the European Social Fund for the postdoctoral contract (APOSTD/2019/149).

Conflicts of interest

There are no conflicts to declare.