Being sessile organisms, plants cannot escape stress and must constantly cope with several environmental challenges, such as light excess and exposure to microorganisms. The enzyme formate dehydrogenase (FDH) catalyzes the oxidation of formate (HCOO-) to carbon dioxide (CO2) along with the reduction of NAD+ to NADH. FDH has been described in the literature as a ‘stress protein’ because its expression is strongly influenced by unfavourable conditions; its induction has been well characterized under abiotic stress, but its role during pathogen attacks has been poorly studied. Therefore, this Ph.D. thesis investigates the response of FDH in Arabidopsis thaliana leaves when plants are exposed to either a vascular pathogen (Xanthomonas campestris pv campestris) or a beneficial rhizobacterium (Pseudomonas simiae WCS417), to further explore the role of FDH in plantbacteria interactions. These investigations have been pursued using a 'hybrid' approach, in which in silico and in vivo strategies have been combined; to promote this type of research approach, researchers should improve the quality of existing data (Manuscript 1) and develop new, user-friendly informatics tools (Manuscript 2). A correlation analysis of Arabidopsis thaliana gene expression data under biotic stress showed that the top correlators of FDH are genes involved in defense responses. Furthermore, in vivo studies using a reporter construct driven by the FDH promoter activity in Arabidopsis thaliana leaves revealed that FDH may have an important role in the early defense response pathways involving hydathodes (specialized pores in plant leaves that secrete excess water in the form of droplets) following Xanthomonas campestris pv campestris infection (Manuscript 3). Pseudomonas simiae WCS417 is likely the best-studied rhizobacterium for its ability to promote an immune response called ‘Induced Systemic Resistance’ (ISR) in Arabidopsis thaliana. FDH promoter activity was rapidly induced in hydathodes of plants colonized by Pseudomonas simiae WCS417 or exposed to the bacterium without direct contact with roots. A total leaf proteome analysis was then performed on wt Col and atfdh1-5 knockout mutants colonized for a short time by the rhizobacterium. Such analysis showed an increase in FDH levels of wt leaves; it also gave indications of the altered leaf metabolic pathways/processes. Changes were mainly related to extrinsic photosystem proteins, stress-responsive proteins, and proteins involved in reactive oxygen species detoxification (Manuscript 4). Notably, ISR partially overlaps with the iron-deficiency response pathway, implicating that iron is not only essential in plant life but also an important element in plant-bacteria interactions (Manuscript 5 and Manuscript 6).
Essendo organismi sessili, le piante non possono fuggire, ma devono costantemente far fronte e diversi stress, come per esempio eccessi di luce o la continua esposizione ai microrganismi. L’enzima formato deidrogenasi (FDH) catalizza l'ossidazione del formato (HCOO-) in anidride carbonica (CO2) insieme alla riduzione di NAD+ in NADH. FDH è stata presentata in letteratura come una ‘proteina di stress’ in quanto la sua espressione è fortemente influenzata dagli stress ambientali; è ben documentata l’induzione in risposta a stress di natura abiotica, mentre il suo ruolo durante gli attacchi patogeni è stato poco studiato. In questa tesi di dottorato si è voluto quindi indagare la risposta di FDH in foglie di Arabidopsis thaliana quando le piante sono esposte a un patogeno del sistema vascolare (Xanthomonas campestris pv campestris) o a un batterio benefico della rizosfera (Pseudomonas simiae WCS417), con l’obiettivo di comprendere meglio il ruolo di FDH nell’interazione pianta-batterio. Si è deciso di utilizzare per le indagini un approccio ‘ibrido’, in quanto si sono usate sia tecniche in silico che in vivo; al fine di favorire questa tipologia di approccio alla ricerca, i ricercatori dovrebbero migliorare la qualità dei dati già esistenti e disponibili (Manoscritto 1) e sviluppare nuovi programmi informatici facili da usare (Manoscritto 2). Un’analisi di correlazione dei dati di espressione genica di Arabidopsis thaliana sotto stress biotico ha mostrato che i principali correlatori di FDH sono geni coinvolti in risposte di difesa. Inoltre, l’esame in vivo dell’attività del promotore di FDH in foglie di Arabidopsis thaliana ha evidenziato come FDH potrebbe avere un ruolo importante nelle prime risposte di difesa degli idatodi (pori specializzati a livello fogliare per secernere l'acqua in eccesso) durante l’infezione da parte di Xanthomonas campestris pv campestris (Manoscritto 3). Molto probabilmente Pseudomonas simiae WCS417 è il batterio della rizosfera più studiato per la sua capacità di promuovere in Arabidopsis thaliana una risposta immunitaria nota come ‘Induced Systemic Resistance’ (ISR). Si è osservata una rapida induzione dell’attività del promotore di FDH negli idatodi di piante colonizzate da Pseudomonas simiae WCS417 o esposte al batterio ma senza un contatto diretto con l’apparato radicale; inoltre, un’analisi globale del proteoma fogliare a seguito di una breve colonizzazione delle radici di piante wt Col e mutanti knockout atfdh1-5 da parte di Pseudomonas simiae WCS417 ha rivelato un aumento dei livelli di FDH nel wt e dato indicazioni sui processi e le vie metaboliche che vengono influenzate a livello delle foglie, in particolare quelli relativi a proteine estrinseche dei fotosistemi, proteine di risposta agli stress, e proteine con funzione di detossificazione di specie reattive dell’ossigeno (Manoscritto 4). Infine, la difesa ISR si sovrappone parzialmente alla risposta della pianta alla ferrocarenza, suggerendo che il ferro è non solo essenziale per la pianta ma che è anche un elemento importante nell’interazione con i microrganismi (Manoscritto 5 e Manoscritto 6).
A 'HYBRID' APPROACH FOR THE CHARACTERIZATION OF POSSIBLE PHYSIOLOGICAL ROLES OF ARABIDOPSIS THALIANA FORMATE DEHYDROGENASE FDH / F. Marzorati ; tutor: P.A. Morandini, I. Murgia ; coordinatore: M. Sugni, M. Guarino. Dipartimento di Scienze e Politiche Ambientali, 2023 Apr 27. 35. ciclo, Anno Accademico 2022.
A 'HYBRID' APPROACH FOR THE CHARACTERIZATION OF POSSIBLE PHYSIOLOGICAL ROLES OF ARABIDOPSIS THALIANA FORMATE DEHYDROGENASE FDH
F. Marzorati
2023
Abstract
Being sessile organisms, plants cannot escape stress and must constantly cope with several environmental challenges, such as light excess and exposure to microorganisms. The enzyme formate dehydrogenase (FDH) catalyzes the oxidation of formate (HCOO-) to carbon dioxide (CO2) along with the reduction of NAD+ to NADH. FDH has been described in the literature as a ‘stress protein’ because its expression is strongly influenced by unfavourable conditions; its induction has been well characterized under abiotic stress, but its role during pathogen attacks has been poorly studied. Therefore, this Ph.D. thesis investigates the response of FDH in Arabidopsis thaliana leaves when plants are exposed to either a vascular pathogen (Xanthomonas campestris pv campestris) or a beneficial rhizobacterium (Pseudomonas simiae WCS417), to further explore the role of FDH in plantbacteria interactions. These investigations have been pursued using a 'hybrid' approach, in which in silico and in vivo strategies have been combined; to promote this type of research approach, researchers should improve the quality of existing data (Manuscript 1) and develop new, user-friendly informatics tools (Manuscript 2). A correlation analysis of Arabidopsis thaliana gene expression data under biotic stress showed that the top correlators of FDH are genes involved in defense responses. Furthermore, in vivo studies using a reporter construct driven by the FDH promoter activity in Arabidopsis thaliana leaves revealed that FDH may have an important role in the early defense response pathways involving hydathodes (specialized pores in plant leaves that secrete excess water in the form of droplets) following Xanthomonas campestris pv campestris infection (Manuscript 3). Pseudomonas simiae WCS417 is likely the best-studied rhizobacterium for its ability to promote an immune response called ‘Induced Systemic Resistance’ (ISR) in Arabidopsis thaliana. FDH promoter activity was rapidly induced in hydathodes of plants colonized by Pseudomonas simiae WCS417 or exposed to the bacterium without direct contact with roots. A total leaf proteome analysis was then performed on wt Col and atfdh1-5 knockout mutants colonized for a short time by the rhizobacterium. Such analysis showed an increase in FDH levels of wt leaves; it also gave indications of the altered leaf metabolic pathways/processes. Changes were mainly related to extrinsic photosystem proteins, stress-responsive proteins, and proteins involved in reactive oxygen species detoxification (Manuscript 4). Notably, ISR partially overlaps with the iron-deficiency response pathway, implicating that iron is not only essential in plant life but also an important element in plant-bacteria interactions (Manuscript 5 and Manuscript 6).File | Dimensione | Formato | |
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phd_unimi_R12733.pdf
Open Access dal 28/04/2024
Descrizione: Tesi di dottorato Francesca Marzorati
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