Leaf apoplastic proteome composition in UV-B treated Arabidopsis thaliana mutants impaired in extracellular glutathione degradation

In plants, environmental perturbations often result in oxidative reactions in the apoplastic space, which are counteracted for by enzymatic and non-enzymatic antioxidative systems, including ascorbate and glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform GGT1 bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with UV-B radiation and studied in redox altered ggt1 mutants. Elevated UV-B has detrimental effects on plant metabolism, plasma membranes representing a major target for ROS generated by this harmful radiation. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in apoplastic protein composition. We then compared the expression changes resulting from the mutation and from the UV-B treatment. Rearrangements occurring in apoplastic protein composition suggest the involvement of hydrogen peroxide, which may ultimately act as a signal. Other important changes related to hormonal effects, cell wall remodeling, and redox activities are also reported. We argue that oxidative stress conditions imposed by UV-B and by disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data shown here are associated with the article from Trentin et al. [1]; protein data have been deposited to the PRIDE database [2] with identifier PXD001807

Gamma-glutamyl cycle in plant's adaptation to environment

This thesis work focusses on the gamma-glutamyl cycle in plants, with the aim to address the physiological significance of this cycle in plant adaptation to the environment. It is composed of three sections, where different approaches have been developed to understand different aspects of the cycle. In consideration that alternative and converging strategies may provide tools for deciphering plant metabolism,two main approaches were adopted: the application of stress conditions, and the use of mutants. In the first work, integrated biochemical, immunocytochemical, and quantitative proteomics analyses were performed in leaves of Arabidopsis thalianaggt1 knockout mutant (lacking apoplastic GGT1 isoform) and itscorresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular-weight range compared to WT. Proteome data showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs, a GSH peroxidase (GPX1), and glyoxylase II, suggesting that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism and photosynthesis, with strong implications for plant’s adaptation to environment. The objective of the second contribution wastounderstand how the ggt1 mutant line responds when it is exposed to an external oxidative stress by UV-B radiation. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition.Results show that, upon UV-B exposure, soluble antioxidants are altered in both genotypes. Rearrangements occur in their apoplastic protein composition, both in the wildtype under UV-B and in the ggt1 mutant in physiological conditions. This suggeststhe involvement of H2O2, which may ultimately act as a signal. I argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Since the gamma-glutamyl transferase operates in the extracellular space, aim of the third contribution was to better investigate the reactions involvingLMW thiols (glutathione,cysteine and cysteinyl-glycine), metals and enzymes related to ROS metabolism in the cell wall. Resultsindicate that LMW thiolsexhibit quenching capacity for reactive oxygen species generated in the apoplastic spaceandpoint to a role for LMW thiols, which are metabolically related to each other in the gamma-glutamyl cycle, in modulating redox reactions in plant cell walls

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for ROS generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform GGT1 bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with UV-B and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of H2O2, which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807

Gamma-glutamyl cycle in plant's adaptation to environment

This thesis work focusses on the gamma-glutamyl cycle in plants, with the aim to address the physiological significance of this cycle in plant adaptation to the environment. It is composed of three sections, where different approaches have been developed to understand different aspects of the cycle. In consideration that alternative and converging strategies may provide tools for deciphering plant metabolism,two main approaches were adopted: the application of stress conditions, and the use of mutants. In the first work, integrated biochemical, immunocytochemical, and quantitative proteomics analyses were performed in leaves of Arabidopsis thalianaggt1 knockout mutant (lacking apoplastic GGT1 isoform) and itscorresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular-weight range compared to WT. Proteome data showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs, a GSH peroxidase (GPX1), and glyoxylase II, suggesting that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism and photosynthesis, with strong implications for plant’s adaptation to environment. The objective of the second contribution wastounderstand how the ggt1 mutant line responds when it is exposed to an external oxidative stress by UV-B radiation. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition.Results show that, upon UV-B exposure, soluble antioxidants are altered in both genotypes. Rearrangements occur in their apoplastic protein composition, both in the wildtype under UV-B and in the ggt1 mutant in physiological conditions. This suggeststhe involvement of H2O2, which may ultimately act as a signal. I argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Since the gamma-glutamyl transferase operates in the extracellular space, aim of the third contribution was to better investigate the reactions involvingLMW thiols (glutathione,cysteine and cysteinyl-glycine), metals and enzymes related to ROS metabolism in the cell wall. Resultsindicate that LMW thiolsexhibit quenching capacity for reactive oxygen species generated in the apoplastic spaceandpoint to a role for LMW thiols, which are metabolically related to each other in the gamma-glutamyl cycle, in modulating redox reactions in plant cell walls.L’argomento della mia tesi di dottorato è stato il ciclo del gamma-glutammile nelle piante con lo scopo generale di investigare il significato fisiologico di questo ciclo nell’adattamento delle piante all’ambiente. Il lavoro è composto da tre contributi sperimentali, nei quali differenti approcci sono stati adottati per capire diversi aspetti del ciclo. Quando si vogliono approfondire le conoscenze per decifrare il metabolismo si possono usare strategie alternative e convergenti, due sono i principali approcci che sono stati adottati: sono stati imposti degli stress ossidativi esterni per valutare la risposta della pianta e si è fatto uso di mutanti. Nel primo lavoro, sono state eseguite analisi biochimiche, immunocitochimiche e proteomiche in foglie di Arabidopsis thaliana del mutante ggt1 (mancante dell’isoforma apoplastica di GGT1) e nel corrispondente wild-type (WT). Comparando le foglie wild-tipe con il mutante ggt1,quest’ultimo presentava un incremento del contenuto di ascorbato e glutatione, anche il GSH apoplastico risultava aumentato e un cambiamento nelle carbonilazioni delle proteinea basso peso molecolare. I dati proteomici evidenziavano che l’interruzione del ciclo del gamma-glutammile nelle foglie del mutante ggt1 era associato con l’induzione di geni codificanti per quattro glutatione-sulfo-transferasi (GSTs), una glutatione perossidasi (GPX1), e la gliossilasi II, suggerendo che la proteina GGT1 ha un ruolo nel redox signaling. Quindi l’interruzione del ciclo del gamma glutammile nel mutante ggt1 porta ad effetti pleiotropici legati alla risposta a stress biotici e abiotici, altera il metabolismo degli antiossidanti, la senescenza, il metabolismo dei carboidrati e la fotosintesi, con forti implicazioni nell’adattamento delle piante all’ambiente. L’obiettivo del secondo contributo era capire come il mutante ggt1 risponde quando è esposto ad un stress ossidativo esterno, è stato scelto di usare la radiazione UV-B. La risposta del mutante ggt1alla radiazione UV-B è stata valutata investigando i cambiamenti nello spazio apoplastico della composizione proteica e del contenuto di glutatione e ascorbato e il loro stato redox. I risultati evidenziano che, l’esposizione all’UV-B, altera gli antiossidanti solubili in entrambi i genotipi. I riarrangiamentiche avvengono nella composizione proteica dell’apoplasto, nel wild-type sottoposto a UV-B e nel mutante ggt1 in condizioni fisiologiche, suggeriscono un coinvolgimento del perossido di idrogeno (H2O2), il quale potrebbe agire come segnale. Questo mi porta a dedurre che le condizioni di stress ossidativo (imposte con l’UV-B) a l’interruzione del ciclo del gamma glutammile,in una certa misura, portano a una simile risposta indotta da stress.Poichè la gamma-glutammil transferase agisce nello spazio extracellulare, scopo del terzo contributo è stato investigare le reazioni che avvengono tra i tioli a basso peso molecolare (glutathione, cisteina e cisteinil-glicina), i metalli e gli enzimi legati al metabolismo dei ROS nella parete cellulare. I risultati indicano che i tioli LMW sono in grado di quenchare le specie attive dell’ossigeno generate nell’ apoplasto e evidenziano un ruolo per i tioli, i quali sono metabolicamente correlati tra loro nel ciclo del gamma-glutammile, nel modulare le reazioni redox nella parete cellulare

Carbon and nitrogen metabolism in barley plants exposed to UV-B radiation

Abstract: The effect of UV-B radiation on FW, leaf and stem length, photosynthetic O2 evolution, levels of carbohydrates and nitrates, and extractable activities of some of the enzymes involved in C and N metabolism was evaluated in barley (Hordeum vulgare L. cv. Express) seedlings during the 9 days following transfer to an UV-B enriched environment. The results show that under our experimental conditions UV-B radiation scarcely affects the photosynthetic competence of barley leaves, expressed as RuBP carboxylase (EC 4.1.1.39) activity, O2 evolution rate and chlorophyll content. Nevertheless, this treatment induced significant alterations of the enzyme activity of nitrate reductase (EC 1.6.6.1) and glutamine synthetase (EC 6.3.1.2), although only after a few days of treatment. The effects were not confined to the exposed tissue, but were detectable also at the root level. In fact, nitrate reductase decreased in response to UV-B in both leaf and root tissue, whereas glutamine synthetase was affected only in the root. In contrast, nitrate content was not influenced by the treatment, neither in root nor in leaf tissue, whilst leaf sucrose diminished in exposed plants only on the last day of treatment