Influenza di diversi fattori abiotici sulla risposta al congelamento di Arabidopsis thaliana

La temperatura rappresenta uno dei più importanti fattori che condizionano la distribuzione geografica delle piante - organismi pecilotermi e sessili - e la produttività delle specie agrarie. Mentre la maggior parte delle specie vegetali di origine tropicale e subtropicale (ivi comprese piante coltivate di grande importanza economica come mais, riso, pomodoro, patata, fagiolo, soia, cotone e molte specie da frutto) incorre in danni da freddo a temperature inferiori ai 10°C, molte piante originarie di climi temperati hanno sviluppato meccanismi che permettono loro non soltanto di mantenere attivi i processi di crescita e di sviluppo in condizioni di gelo, ma altresì di sopportare successivi eventi di congelamento. La risposta della pianta agli stress da basse temperature è un carattere quantitativo (multigenico), influenzato tanto dalle caratteristiche dello stress ambientale (temperatura, durata dell'esposizione) quanto dalla capacità di acclimatazione dell'organismo (fenomeno complesso e inducibile, risultante nell'acquisizione di tolleranza al congelamento in seguito a un periodo di temperature basse ma superiori a quelle di congelamento). Un'intensa e prolungata attività di ricerca ha evidenziato che stress ambientali il cui effetto primario consista nella disidratazione cellulare, come gli stress idrici, la salinità, gli stress osmotici e il congelamento medesimo, conducono spesso a cambiamenti simili nel metabolismo della pianta e nell'espressione genica, indicando l'esistenza di estese interazioni nelle rispettive vie di percezione e risposta. L'obiettivo del presente lavoro è valutare, dunque, se fattori abiotici contraddistinti da una componente di disidratazione siano in grado di esercitare un effetto sulla risposta al congelamento di piante trattate e se, in caso affermativo, tale effetto abbia carattere protettivo oppure sia di detrimento; a questo scopo è stata scelta la specie modello Arabidopsis thaliana, in ragione della sua versatilità e dell'ampia disponibilità di risorse molecolari e bioinformatiche ad essa relative. Il comportamento di piante sottoposte a stress da salinità, da siccità e all'applicazione di ABA esogeno (che mima, a livello cellulare, gli stress osmotici e da disidratazione) è stato comparato con quello di piante mantenute in condizioni di controllo e di piante acclimatate, in termini sia di sopravvivenza al congelamento che di tolleranza al medesimo, per la quale sono stati impiegati come parametri la stabilità delle membrane, il contenuto in pigmenti fogliari e la concentrazione fogliare di zuccheri liberi. In seguito, l'esistenza di cross-talk fra il congelamento e i fattori abiotici e la loro ampiezza sono state valutate attraverso l'esame dei profili proteici, prodotti mediante elettroforesi bidimensionale. I risultati ottenuti hanno consentito di individuare differenze significative fra i controlli e alcuni dei trattamenti effettuati, nonché di identificare spot proteici differenzialmente espressi all'interno dei profili corrispondenti. Dall'attribuzione degli spot d'interesse, che può essere raggiunta mediante spettrometria di massa, sarebbe possibile ricavare ulteriori informazioni in merito al ruolo funzionale dei prodotti individuati nella difesa della pianta dallo stress da congelamento

Synthetic biology of hypoxia.

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Functional Balancing of the Hypoxia Regulators RAP2.12 and HRA1 Takes Place in vivo in Arabidopsis thaliana Plants

Plants are known to respond to variations in cellular oxygen availability and distribution by quickly adapting the transcription rate of a number of genes, generally associated to improved energy usage pathways, oxygen homeostasis and protection from harmful products of anaerobic metabolism. In terrestrial plants, such coordinated gene expression program is promoted by a conserved subfamily of ethylene responsive transcription factors called ERF-VII, which act as master activators of hypoxic gene transcription. Their abundance is directly regulated by oxygen through a mechanism of targeted proteolysis present under aerobic conditions, which is triggered by ERF-VII protein oxidation. Beside this, in Arabidopsis thaliana, the activity of the ERF-VII factor RAP2.12 has been shown to be restrained and made transient by the hypoxia-inducible transcription factor HRA1. This feedback mechanism has been proposed to modulate ERF-VII activity in the plant under fluctuating hypoxia, thereby enhancing the flexibility of the response. So far, functional balancing between RAP2.12 and HRA1 has been assessed in isolated leaf protoplasts, resulting in an inverse relationship between HRA1 amount and activation of RAP2.12 target promoters. In the present work, we showed that HRA1 is effective in balancing RAP2.12 activity in whole arabidopsis plants. Examination of a segregating population, generated from RAP2.12 and HRA1 over-expressing plants, led to the first quantitative proof that, over a range of either transgene expression levels, HRA1 counteracts the phenotypic and transcriptional effects of RAP2.12. This report supports the occurrence of fine-tuned regulation of the hypoxic response under physiological growth conditions

The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2.12, RAP2.2 and RAP2.3

The ethylene response factor VII (ERF-VII) transcription factor RELATED TO APETALA2.12 (RAP2.12) was previously identified as an activator of the ALCOHOL DEHYDROGENASE1 promoter::luciferase (ADH1-LUC) reporter gene. Here we show that overexpression of RAP2.12 and its homologues RAP2.2 and RAP2.3 sustains ABA-mediated activation of ADH1 and activates hypoxia marker genes under both anoxic and normoxic conditions. Inducible expression of all three RAP2s conferred tolerance to anoxia, oxidative and osmotic stresses, and enhanced the sensitivity to abscisic acid (ABA). Consistently, the rap2.12-2 rap2.3-1 double mutant showed hypersensitivity to both submergence and osmotic stress. These findings suggest that the three ERF-VII-type transcription factors play roles in tolerance to multiple stresses that sequentially occur during and after submergence in Arabidopsis. Oxygen-dependent degradation of RAP2.12 was previously shown to be mediated by the N-end rule pathway. During submergence the RAP2.12, RAP2.2 and RAP2.3 are stabilized and accumulates in the nucleus affecting the transcription of stress response genes. We conclude that the stabilized RAP2 transcription factors can prolong the ABA-mediated activation of a subset of osmotic responsive genes (e.g. ADH1). We also show that RAP2.12 protein level is affected by the REALLY INTERESTING GENE (RING) domain containing SEVEN IN ABSENTIA of Arabidopsis thaliana 2 (SINAT2). Silencing of SINAT1/2 genes leads to enhanced RAP2.12 abundance independently of the presence or absence of its N-terminal degron. Taken together, our results suggest that RAP2.12 and its homologues RAP2.2 and RAP2.3 act redundantly in multiple stress responses. Alternative protein degradation pathways may provide inputs to the RAP2 transcription factors for the distinct stresses

Tuberomics: a molecular profiling for the adaption of edible fungi (Tuber magnatum Pico) to different natural environments

Background: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. Results: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. Conclusions: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups. A-type PCOs are conserved across all plant species and are generally unaffected at the messenger RNA level by oxygen availability. Instead, B-type PCOs appeared in spermatophytes to acquire transcriptional regulation in response to hypoxia. The inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperms. Therefore, while it is possible to distinguish two clades within the PCO family, we conclude that they all contribute to restrain the anaerobic transcriptional programme in normoxic conditions and together generate a molecular switch to toggle the hypoxic response

Molecular characterization of At3g10040, a low oxygen-responsive gene of Arabidopsis thaliana

In this work, the functional characterization of At3g10040, a trihelix TF-coding gene of Arabidopsis thaliana with unassigned molecular function, has been carried out. Transcriptomic analyses performed in the past years have led to the identification of a core of low oxygen-responsive genes in arabidopsis, among which At3g10040 is found as one of the most strongly and early up-regulated. Over-expression of the gene in arabidopsis causes the attenuation of the transcriptional response to anoxia, but nevertheless proves to be beneficial for the plant stress tolerance. Although At3g10040 promoter and leader sequence are able to drive the anoxic transcription of a downstream reporter, translation is mainly post-anoxic, suggesting that At3g10040 might be implied in gene repression during the post-stress recovery. We put forward a role for At3g10040 in dampening the fermentative metabolism, in a cellular strategy aimed at optimizing the rates of energy production after a low oxygen stress. The presence of a submergence-responsive At3g10040 ortholog in rice outlines the possibility of a conserved strategy in plant post-hypoxic responses