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Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.

Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.

Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Plants sense light intensity, quality and direction through a group of photoreceptors to modulate their growth and development. One family of photoreceptor is called phytochromes (phys) that perceives red and far red light. Phys transduce light signals via a sub-family of the basic Helix-Loop-Helix (bHLH) transcription factors called Phytochrome Interacting Factors (PIFs). PIFs function as negative regulators in the phy-mediated light signaling pathways. In darkness, PIFs regulate downstream gene expressions to inhibit photomorphogenesis. Upon light exposure, PIFs are phosphorylated and poly-ubiquitylated prior to their rapid degradation through the 26S proteasome pathway. One of the PIFs, PIF1, has the highest affinity for both phyA and phyB and also displayed the fastest degradation kinetics under both red and far red light. Here we showed that PIF1 directly and indirectly regulates key genes involved in chlorophyll biosynthesis to optimize the greening process in Arabidopsis. PIF1 binds to a G-box (CACGTG) DNA sequence element present in its direct target genes (e.g., protochlorophyllide oxidoreductase C, PORC) in darkness and regulates their expression. Structure-function studies revealed two separate regions called APB and APA necessary for binding to phyB and phyA, respectively, located at the amino-terminus and a novel phosphorylation site at the carboxy-terminus of PIF1. Both amino- and carboxy-terminal regions are necessary for the light-induced degradation of PIF1. However, the DNA binding is not necessary for the light-induced degradation of PIF1. Using a targeted systems biology approach, we identified new factors, HECATE proteins that promote photomorphogenesis by negatively regulating the function of PIF1. Moreover, we employed an unbiased genetic screening using luciferase imaging system to identify new mutants defective in the light-induced degradation of PIF1. The cloning and characterization of these mutants will help identify the factors, such as the kinase and E3 ligase, responsible for the light-induced degradation of PIF1. Taken together, these data revealed detail mechanisms of how PIF1 negatively regulates photomorphogenesis and how light induces rapid degradation of PIF1 to promote photomorphogenesis. / text

Phytochromes (Phys) are biliproteins that regulate light responses in plants, fungi, and microorganisms through photoconversion between a dark state and a photoproduct. Thermal reversion of the photoproduct is an intrinsic property of all Phys, typically occurring on a timescale of seconds to days. Despite methodological advances, the structural and spectroscopic determination of short-lived photoproducts has proven challenging. We herein present an innovative approach for photoproduct stabilisation by incorporating the protein into trehalose glasses (TGs). The resulting Phy–trehalose matrices were investigated by UV/Vis absorption and solid-state NMR spectroscopies. Our results demonstrate that the TGs strongly inhibit thermal reversion of the incorporated Phy proteins for periods as long as several weeks at room temperature (RT), during which the proteins fully sustain their native structures and spectral and biochemical properties. This sample preparation approach is beneficial for revealing bona fide structure/ function relationships of short-lived photoproducts that are otherwise not accessible, thus paving the way towards a deeper molecular understanding of the diversified spectral properties of Phys. Our results also provide new insights into the molecular mechanism of trehalose bioprotection.

O desestiolamento vegetal envolve a conversão de etioplastos em cloroplastos maduros e plenamente funcionais, sendo desencadeado pela luz através de um processo multifacetado que se baseia em redes de sinalização endógenas diversificadas e altamente coordenadas. Acredita-se que hormônios vegetais ou outras moléculas sinalizadoras, tais como o radical livre óxido nítrico (NO), desempenham papel importante na regulação desse conjunto de respostas fotomorfogênicas. No presente estudo, buscamos investigar, de forma integrada, a influência do NO, do etileno e das auxinas na indução do acúmulo de pigmentos fotossintéticos e desenvolvimentos dos cloroplastos desencadeados pela luz em plântulas de tomateiro (Solanum lycopersicum). Por meio da determinação do padrão temporal de acúmulo de pigmentos fotossintéticos, diferenciação de etioplastos em cloroplastos, flutuações nos teores endógenos de NO e na atividade e estado de ativação da nitrato redutase (NR) em plântulas do tipo selvagem (cultivar Micro-Tom, MT) e de mutantes fotomorfogênicos (áurea and high pigment 1) mantidas sob escuro contínuo ou expostas às luzes monocromáticas vermelha e azul, pudemos constatar uma clara correlação positiva entre a produção de NO via NR e a indução do acúmulo de pigmentos e desenvolvimento dos cloroplastos em resposta à luz. Dando suporte à importância da NR como fonte biossintética de NO nas plântulas de tomateiro em processo de desestiolamento, constatou-se que as diferentes estratégias empregadas com o intuito de inibir a indução da atividade dessa enzima em resposta à luz resultaram em reduções consideráveis na produção endógena de NO. De modo interessante, tratamentos com NO estimularam o acúmulo de pigmentos e a diferenciação plastidial nas células cotiledonares do mutante áurea sob luz vermelha, indicando, portanto, que essa molécula sinalizadora seria capaz de complementar a deficiência partial na percepção da luz vermelha característica desse mutante deficiente em fitocromos. Em paralelo, um antagonismo mútuo entre o NO e o etileno foi evidenciado por meio de uma série de constatações. (i) O acúmulo de pigmentos e diferenciação de cloroplastos induzidos nas plântulas de tomateiro em resposta às luzes vermelha e azul coincidiram temporalmente com um aumento e diminuição nas emissões de NO e etileno, respectivamente. (ii) Enquanto o NO se mostrou estimulatório ao acúmulo de pigmentos, tratamentos com etileno gasoso ou com o seu precursor (o ácido 1-aminociclopropano-1-carboxílico, ACC) drasticamente inibiram o acúmulo de pigmentos em resposta às luzes vermelha ou azul. (iii) Plântulas em processo de desestiolamento tratadas com etileno ou ACC apresentaram níveis reduzidos de NO, ao passo que plântulas do mutante com baixa sensibilidade ao etileno Never ripe (Nr) exibiram teores de NO endógeno significativamente aumentados. (iv) Plântulas de Nr em processo de desestiolamento apresentaram incrementos consideráveis tanto na atividade total quanto no estado de ativação da NR, uma enzima produtora de NO. (v) NO exógeno reduziu drasticamente a emissão de etileno em plântulas do mutante áurea mantidas sob luz vermelha. Em contrapartida, diversas evidências revelaram um sinergismo mútuo entre auxinas e NO durante o processo de destiolamento em plântulas de tomateiro. (i) O acúmulo de NO em resposta à luz coincidiu com um aumento na ativação do promotor sintético responsivo à auxinas DR5 em plantas de MT expostas às luzes vermelha ou azul. (ii) A suplementação com NO gasoso reestabeleceu a reduzida ativação do promotor DR5 observada em plântulas de áurea sob luz vermelha. (iii) Os teores endógenos de NO foram drasticamente aumentados e diminuídos em plântulas do mutante com baixa sensibilidade à auxinas (diageotropica) e no mutante hipersensível à auxinas (entire), respectivamente. Em conjunto, os dados obtidos parecem indicar que durante a indução do acúmulo de pigmentos fotossintéticos e diferenciação de cloroplastos em plântulas estioladas de tomateiro as interações NO-etileno e NO-auxinas seriam controladas via mecanismos regulatórios de retroalimentação positiva e negativa, respectivamente; e, assim, tais relações hormonais desempenhariam papel importante na coordenação da transição dessas plântulas do estado estiolado para o desenvolvimento fotomorfogênico / The transition from etiolated to green seedlings involves the conversion of etioplasts into mature, functional chloroplasts via a multifaceted light-driven process comprising multiple and tightly coordinated endogenous signaling networks. Plant hormones and other signaling molecules, such as the free radical nitric oxide (NO), are believed to play important roles in controlling the acquisition of these photomorphogenic traits. In the present study, we investigated, in an integrated way, the influence of NO, ethylene and auxins on the light-evoked greening and chloroplast development in tomato (Solanum lycopersicum) seedlings. By determining the time course of photosynthetic pigments accumulation, etioplast-to-chloroplast differentiation, fluctuations in endogenous NO content and in nitrate reductase (NR) total activity and activation state in wild type (Micro-Tom cultivar, MT) and in photomorphogenic mutants (aurea and high pigment 1) seedlings maintained under continuous darkness or exposed to monochromatic red (RL) or blue light (BL), we evidenced a clearly positive correlation between the NO production via NR and the light-induced cotyledon greening and chloroplast maturation. Supporting a role for NR as an important biosynthetic source of NO in de- etiolating tomato seedlings, different strategies employed to inhibit the light-evoked increment in the activity of this enzyme successfully reduced the endogenous NO production. Interestingly, exogenous NO stimulated greening and chloroplast differentiation in cotyledon cells of áurea seedlings maintained under RL, thereby indicating that this signaling molecule might complement the partial deficiency in RL perception characteristic of this phytochrome-deficient mutant. In parallel, a mutual antagonism between NO and ethylene was evidenced by a number of findings. (i) RL- or BL-induced greening and chloroplast differentiation in tomato seedlings temporally coincided with increases and decreases in NO and ethylene emission, respectively. (ii) Whereas NO stimulated cotyledon greening, treatments with gaseous ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid, ACC) severally impaired either RL- or BL-induced greening in MT. (iii) Ethylene- or ACC-treated de-etiolating seedlings presented significantly lower NO levels whereas the ethylene-insensitive Never ripe (Nr) mutant exhibited increased endogenous NO content. (iv) De-etiolating Nr seedlings exhibited increased total activity and activation state of the NO-generating enzyme NR. (v) Exogenous NO drastically reduced ethylene emission in au seedlings maintained under RL. On the other hand, a series of evidence indicated a mutual synergism between auxins and NO in de-etiolating tomato seedlings. (i) The light-induced NO accumulation coincided with an increased activation of the synthetic auxin-responsive promoter DR5 in both RL- and BL-exposed MT seedlings. (ii) Exogenous NO completely rescued the reduced activation of the DR5 promoter observed in au seedlings under RL. (iii) Endogenous NO was drastically decreased and increased in de-etiolating seedlings of auxin-insensitive (diageotropica) and auxin-hypersensitive (entire) tomato mutants, respectively. Taken together, these data reveal that negative and positive feedback regulatory loops orchestrate ethylene-NO and auxin-NO interactions during the light-triggered cotyledon greening and chloroplast differentiation in de-etiolating tomato seedlings, reinforcing the importance of these signaling molecules during the coordination of seedling transition from the etiolated state to photomorphogenic growth

O desestiolamento vegetal envolve a conversão de etioplastos em cloroplastos maduros e plenamente funcionais, sendo desencadeado pela luz através de um processo multifacetado que se baseia em redes de sinalização endógenas diversificadas e altamente coordenadas. Acredita-se que hormônios vegetais ou outras moléculas sinalizadoras, tais como o radical livre óxido nítrico (NO), desempenham papel importante na regulação desse conjunto de respostas fotomorfogênicas. No presente estudo, buscamos investigar, de forma integrada, a influência do NO, do etileno e das auxinas na indução do acúmulo de pigmentos fotossintéticos e desenvolvimentos dos cloroplastos desencadeados pela luz em plântulas de tomateiro (Solanum lycopersicum). Por meio da determinação do padrão temporal de acúmulo de pigmentos fotossintéticos, diferenciação de etioplastos em cloroplastos, flutuações nos teores endógenos de NO e na atividade e estado de ativação da nitrato redutase (NR) em plântulas do tipo selvagem (cultivar Micro-Tom, MT) e de mutantes fotomorfogênicos (áurea and high pigment 1) mantidas sob escuro contínuo ou expostas às luzes monocromáticas vermelha e azul, pudemos constatar uma clara correlação positiva entre a produção de NO via NR e a indução do acúmulo de pigmentos e desenvolvimento dos cloroplastos em resposta à luz. Dando suporte à importância da NR como fonte biossintética de NO nas plântulas de tomateiro em processo de desestiolamento, constatou-se que as diferentes estratégias empregadas com o intuito de inibir a indução da atividade dessa enzima em resposta à luz resultaram em reduções consideráveis na produção endógena de NO. De modo interessante, tratamentos com NO estimularam o acúmulo de pigmentos e a diferenciação plastidial nas células cotiledonares do mutante áurea sob luz vermelha, indicando, portanto, que essa molécula sinalizadora seria capaz de complementar a deficiência partial na percepção da luz vermelha característica desse mutante deficiente em fitocromos. Em paralelo, um antagonismo mútuo entre o NO e o etileno foi evidenciado por meio de uma série de constatações. (i) O acúmulo de pigmentos e diferenciação de cloroplastos induzidos nas plântulas de tomateiro em resposta às luzes vermelha e azul coincidiram temporalmente com um aumento e diminuição nas emissões de NO e etileno, respectivamente. (ii) Enquanto o NO se mostrou estimulatório ao acúmulo de pigmentos, tratamentos com etileno gasoso ou com o seu precursor (o ácido 1-aminociclopropano-1-carboxílico, ACC) drasticamente inibiram o acúmulo de pigmentos em resposta às luzes vermelha ou azul. (iii) Plântulas em processo de desestiolamento tratadas com etileno ou ACC apresentaram níveis reduzidos de NO, ao passo que plântulas do mutante com baixa sensibilidade ao etileno Never ripe (Nr) exibiram teores de NO endógeno significativamente aumentados. (iv) Plântulas de Nr em processo de desestiolamento apresentaram incrementos consideráveis tanto na atividade total quanto no estado de ativação da NR, uma enzima produtora de NO. (v) NO exógeno reduziu drasticamente a emissão de etileno em plântulas do mutante áurea mantidas sob luz vermelha. Em contrapartida, diversas evidências revelaram um sinergismo mútuo entre auxinas e NO durante o processo de destiolamento em plântulas de tomateiro. (i) O acúmulo de NO em resposta à luz coincidiu com um aumento na ativação do promotor sintético responsivo à auxinas DR5 em plantas de MT expostas às luzes vermelha ou azul. (ii) A suplementação com NO gasoso reestabeleceu a reduzida ativação do promotor DR5 observada em plântulas de áurea sob luz vermelha. (iii) Os teores endógenos de NO foram drasticamente aumentados e diminuídos em plântulas do mutante com baixa sensibilidade à auxinas (diageotropica) e no mutante hipersensível à auxinas (entire), respectivamente. Em conjunto, os dados obtidos parecem indicar que durante a indução do acúmulo de pigmentos fotossintéticos e diferenciação de cloroplastos em plântulas estioladas de tomateiro as interações NO-etileno e NO-auxinas seriam controladas via mecanismos regulatórios de retroalimentação positiva e negativa, respectivamente; e, assim, tais relações hormonais desempenhariam papel importante na coordenação da transição dessas plântulas do estado estiolado para o desenvolvimento fotomorfogênico / The transition from etiolated to green seedlings involves the conversion of etioplasts into mature, functional chloroplasts via a multifaceted light-driven process comprising multiple and tightly coordinated endogenous signaling networks. Plant hormones and other signaling molecules, such as the free radical nitric oxide (NO), are believed to play important roles in controlling the acquisition of these photomorphogenic traits. In the present study, we investigated, in an integrated way, the influence of NO, ethylene and auxins on the light-evoked greening and chloroplast development in tomato (Solanum lycopersicum) seedlings. By determining the time course of photosynthetic pigments accumulation, etioplast-to-chloroplast differentiation, fluctuations in endogenous NO content and in nitrate reductase (NR) total activity and activation state in wild type (Micro-Tom cultivar, MT) and in photomorphogenic mutants (aurea and high pigment 1) seedlings maintained under continuous darkness or exposed to monochromatic red (RL) or blue light (BL), we evidenced a clearly positive correlation between the NO production via NR and the light-induced cotyledon greening and chloroplast maturation. Supporting a role for NR as an important biosynthetic source of NO in de- etiolating tomato seedlings, different strategies employed to inhibit the light-evoked increment in the activity of this enzyme successfully reduced the endogenous NO production. Interestingly, exogenous NO stimulated greening and chloroplast differentiation in cotyledon cells of áurea seedlings maintained under RL, thereby indicating that this signaling molecule might complement the partial deficiency in RL perception characteristic of this phytochrome-deficient mutant. In parallel, a mutual antagonism between NO and ethylene was evidenced by a number of findings. (i) RL- or BL-induced greening and chloroplast differentiation in tomato seedlings temporally coincided with increases and decreases in NO and ethylene emission, respectively. (ii) Whereas NO stimulated cotyledon greening, treatments with gaseous ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid, ACC) severally impaired either RL- or BL-induced greening in MT. (iii) Ethylene- or ACC-treated de-etiolating seedlings presented significantly lower NO levels whereas the ethylene-insensitive Never ripe (Nr) mutant exhibited increased endogenous NO content. (iv) De-etiolating Nr seedlings exhibited increased total activity and activation state of the NO-generating enzyme NR. (v) Exogenous NO drastically reduced ethylene emission in au seedlings maintained under RL. On the other hand, a series of evidence indicated a mutual synergism between auxins and NO in de-etiolating tomato seedlings. (i) The light-induced NO accumulation coincided with an increased activation of the synthetic auxin-responsive promoter DR5 in both RL- and BL-exposed MT seedlings. (ii) Exogenous NO completely rescued the reduced activation of the DR5 promoter observed in au seedlings under RL. (iii) Endogenous NO was drastically decreased and increased in de-etiolating seedlings of auxin-insensitive (diageotropica) and auxin-hypersensitive (entire) tomato mutants, respectively. Taken together, these data reveal that negative and positive feedback regulatory loops orchestrate ethylene-NO and auxin-NO interactions during the light-triggered cotyledon greening and chloroplast differentiation in de-etiolating tomato seedlings, reinforcing the importance of these signaling molecules during the coordination of seedling transition from the etiolated state to photomorphogenic growth

Au cours de l’évolution, les plantes ont développé des mécanismes pour percevoir et s'adapter aux conditions de stress. Les formes actives de l'oxygène (FAO) sont des facteurs importants de l'état redox cellulaire et sont impliquées dans ces réponses. Le peroxyde d'hydrogène (H2O2), une FAO majeure des voies de signalisation oxydative, peut être produit rapidement dans la photorespiration. Chez Arabidopsis, le H2O2 produit dans la photorespiration est métabolisé notamment par la CATALASE2 (CAT2). Dans le contexte du mutant cat2 déficient pour cette catalase, les réponses au stress oxydatif induit par la production conditionnelle du H2O2 sont fortement dépendante de la photopériode. En particulier, la formation de lésions, accompagnée de réponses similaires à celles d' attaques pathogènes, sont spécifiques des conditions de culture en jours longs (JL). Ces effets ne sont pas observés en jours courts (JC) malgré un stress oxydant qui semble être aussi prononcé qu’en JL. Une approche transcriptomique globale a été utilisée pour explorer les patterns d’expression génique associées à ces effets. Elle a permis de mettre en évidence des interactions entre photopériode et H2O2 ou entre photopériode et CO2. En particulier, la majorité des gènes répondant à l' H2O2 dans le mutant cat2 sont induits lorsque les plantes sont cultivées en JC alors que un plus petit nombre sont induits par l’ H2O2 spécifiquement en JL. De façon générale, ces analyses ont mis en évidence des relations étroites entre les ressources carbonées, la lumière et l'état redox cellulaire dans les réponses aux changements environnementaux. Un gène induit par le H2O2 spécifiquement en JL, l’AZELAIC ACID INDUCED 1 (AZI1), a été sélectionné pour des analyses fonctionnelles à l’aide d’approches génétique, biochimique et transcriptomique. L’analyse de mutants cat2 azi1 a révélé que AZI1 ne semble pas jouer un rôle majeur dans les réponses des plantes à un stress oxydatif durable. Cependant, ce gène semble jouer un rôle important lorsque le stress oxydatif est déclenchée de façon abrupte par le transfert des plantes de conditions de culture en fort CO2 vers l'air ambiant. De plus, cette étude montre que la communication de feuille à feuille est impliquée dans la régulation de l'expansion de la mort cellulaire en réponse a l'H2O2 issue de la photorespiration. Dans la régulation de l'expansion des lésions, nous proposons que AZI1 agirait d'une part localement pour induire la mort cellulaire et d'autre en inhibant la mort cellulaire d'une façon systémique. Dans des fonds génétiques sauvage Col-0 ou mutant cat2, l’analyse comparative de mutants d'insertion ADN-T pour les principaux photochromes (phyA , phyB) et cryptochromes (cry1, cry2) a permis d'étudier les interactions entre les stress et les fonctions des photorécepteurs. Il est apparu que, la mutation des gènes PHY comme CRY conduit a une stimulation de l’accumulation de glutathion H2O2 dépendante. En revanche, dans le fond génétique cat2 contrairement à la perte des fonctions PHY, la mutation des gènes cry conduit a une modulation du profil transcritomique induit par l’ H2O2. De plus, un criblage de conditions de stress sur les simples mutants cry a révélé une plus forte sensibilité de ces génotypes au stress osmotique, a l’ H2O2 et au paraquat. Globalement, ces données indiquent que l’ensemble des photorécepteurs et plus particulièrement les cryptochromes peuvent jouer un rôle dans la réponse à l’ H2O2 intracellulaire suggérant ainsi l’existence d’un réseau complexe permettant l’intégration de conditions environnementales et la détermination de réponses appropriées au stress. / During evolution, plants have developed mechanisms to perceive and respond to stress conditions. Reactive oxygen species (ROS) are important components of cell redox state that have been implicated in these responses. H2O2, an important ROS molecule in oxidative signalling, can be produced rapidly in photorespiration. In Arabidopsis, photorespiratory H2O2 is notably metabolized by CATALASE2 (CAT2). Responses to oxidative stress induced conditionally by photorespiratory H2O2 in the catalase-deficient mutant, cat2, are highly determined by growth daylength. In particular, lesion formation, accompanied by induction of a range of pathogenesis responses, is specific to the long day (LD) photoperiod: these responses are not observed in short days (SD), even though oxidative stress seems to be as marked as in LD. A whole-genome transcriptomics approach was used to explore gene expression patterns underlying these effects, and identified interactions between daylength and H2O2 and between daylength and CO2. In particular, the majority of H2O2-responsive genes in cat2 were up-regulated more strongly in SD air, though a subset of H2O2-induced genes showed a LD-specific response. Overall, this analysis indicates close networking between carbon status, light, and redox state in environmental responses. The most strongly H2O2-induced gene in LD was azelaic acid induced 1 (AZI1) and this gene was chosen for functional analysis using a genetic, biochemical and transcript profiling approach. Analysis of cat2 azi1 mutants revealed that AZI1 does not seem to play an important role in the plant response to sustained, continuous oxidative stress, but is influential when oxidative stress is abruptly induced, in this case, by transferring plants from high CO2 to air. Moreover, this study provided evidence that leaf-to-leaf communication is involved in regulating cell death spread in response to photorespiratory H2O2. In the regulation of this lesion spread, it is proposed that AZI1 acts both locally to promote cell death as well as systemically to inhibit it. Using a comparative analysis of T-DNA insertion mutants for the major phytochromes (phyA, phyB) and cryptochromes (cry1, cry2) introduced into the Col-0 or cat2 background, interactions between stress and photoreceptor function were analyzed. A stimulatory effect of both phy and cry mutations on H2O2-triggered glutathione accumulation was apparent. In contrast to loss of PHY function, both cry mutations modulated daylength-dependent H2O2-triggered transcriptome profiles in cat2. In addition, stress screening of single cry mutants revealed effects on osmotic, H2O2 and paraquat sensitivity. Overall, these data show that both kinds of photoreceptor, but particularly cryptochromes, can play a role in the response to intracellular H2O2, suggesting that there is an intricate network allowing integration of environmental information to determine appropriate responses to stress.

Via la photosynthèse et la photomorphogenèse, la lumière contrôle fortement la ramification. Le rosier (Rosa hybrida L.) présente par exemple une inhibition complète du débourrement à l’obscurité. Mieux comprendre le mode d’action de la lumière offrira des possibilités nouvelles à l’horticulture. Peu de choses sont connues sur la signalisation de la lumière au cours du débourrement. Notamment, les rôles de chacun des photorécepteurs et des cytokinines (CK), hormones promotrices du débourrement, dans la transduction du signal lumineux n’ont pas été examinés. Dans ce travail, des apports exogènes et des dosages de CK à l’obscurité montrent que le photocontrôle du débourrement passe par la photorégulation des CK. La lumière contrôle la néosynthèse et le transport des CK depuis l’entre-nœud vers le bourgeon, et inhibe leur dégradation. Les CK néoformées initient le débourrement car elles régulent un ensemble de gènes majeurs du débourrement (signalisation des strigolactones, métabolisme et transport de l’auxine et des sucres, division et expansion cellulaires). Afin d’identifier les photorécepteurs impliqués dans ce processus, des études ont été menées chez le pois (Pisum sativum L.). Il en ressort que le profil de débourrement chez le pois est sous photocontrôle : basitone à la lumière, ce profil devient acrotone à l’obscurité ou suite aux simples et doubles mutations de phyA, phyB et cry1. Ces trois photorécepteurs jouent donc un rôle primordial sur les corrélations inhibitrices entre bourgeons. Des apports de CK permettent de restaurer le profil basitone du sauvage chez le mutant phyB, ce qui suggère que la signalisation de la lumière vers les CK passe par phyB. / Through photosynthesis and photomorphogenesis, light highly controls plant branching. Roses (Rosa hybrida L.) exhibit for example a strong inhibition of bud outgrowth under darkness. Better understanding the action of light in branching will give new opportunities to horticulturists. Yet, little is known about the light signalling pathway during bud outgrowth. In particular, the involvements of each photoreceptor and of cytokinins (CK), plant hormones acting as promoters of bud outgrowth, in the transduction of the light signal during bud outgrowth have not been assessed. In this work, exogenous applications and quantification of CK under darkness show that the photocontrol of bud outgrowth goes through the photo-regulation of CK. Light controls CK neo-synthesis and transport from the internode toward the bud, and inhibits their degradation. The neo-synthesised CK initiate bud outgrowth by regulating a set of major genes involved in bud outgrowth (strigolactones signalling, metabolisms and transports of auxin and of sugar, cell division and expansion). In order to identify the photoreceptors that are involved in this process, we studied pea (Pisum sativum L.) mutants. Our research indicate that the bud outgrowth profile along the stem is under photocontrol: being basitonic under white light, this profile becomes acrotonic under darkness or after mutations of phyA, phyB and cry1. These three photoreceptors thus play a major role in the control of the correlative inhibitions between buds. Since applications of CK allowed to restore the wild-type basitonic profile in the phyB mutant, this suggests that the light signalling pathway towards CK involves phyB.