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Effects of enod40 overexpression in non legume plants
Full text linkIl gene ENOD40 è una nodulina precoce ed è indotto durante i primi stadi di formazione del nodulo radicale in risposta alle interazioni tra le leguminose ed i batteri simbionti del genere Rhizobia.
Omologhi del gene ENOD40 sono stati identificati in diverse specie e la sua espressione, non unicamente correlata alla formazione del nodulo, è stata osservata in tessuti giovani e meristematici.
Una caratteristica che accomuna i geni ENOD40 è l’assenza di un lunga open reading frame (ORF); al contrario, molte piccole ORF sono generalmente presenti nei trascritti. Il gene contiene due regioni altamente conservate chiamate box1 e box2. Tra le diverse specie è conservata l’ORF (ORF1) del box1, che sembra codificare per un putativo peptide di 10-13 amminoacidi. Inoltre, il gene contiene regioni corrispondenti a strutture conservate a livello del trascritto. Sei domini sono stati individuati nel mRNA del gene e due di questi domini sono fortemente conservati tra le leguminose e le non leguminose.
Nonostante decenni di ricerche, il ruolo del gene ENOD40 non è stato finora completamente chiarito. La natura biologica del gene è tuttora in discussione, infatti se l’attività biologica del gene dipenda dall’ RNA o da entrambi è ancora da chiarire.
I due principali obiettivi del mio progetto di ricerca sono: da una parte, indagare la possibile presenza del putativo peptide codificato dal box1 utilizzando cellule BY-2 che overesprimono il gene e dall’altra, studiare il ruolo del gene ENOD40 in piante non leguminose, utilizzando Arabidopsis thaliana.
Nella prima parte del lavoro è stata messa appunto una procedura di purificazione per cercare il putativo peptide in cellule BY-2 che overesprimevano il gene ENOD40 di tabacco. Fin ad ora il putativo peptide non è mai stato trovato in vivo; è stato però suggerito da diverse osservazioni che il gene potrebbe, almeno in parte, agire attraverso il peptide codificato dall’ORF1.
La procedura messa appunto consiste in un cut-off iniziale, seguita da cromatografia a scambio ionico, estrazione di cambio solido, HPLC-DAD e spettrometria di massa (LC-ESI-MS e MALDI-TOF). Purtroppo, nonostante i diversi tentativi per mettere appunto la procedura di purificazione e le diverse tecniche utilizzate per l'analisi delle frazioni putativamente peptide-arricchite, solo l’analisi MALDI-TOF PSD ha dato un primo indizio sulla possibile presenza del peptide in cellule BY-2 che overesprimevano il gene ENOD40.
Nella seconda parte del lavoro, il possibile ruolo del gene è stato indagato mediante l’analisi metabolomica e trascrizionale in piante di Arabidopsis che overesprimevano il gene ENOD40 di soia.
I profili metabolici e trascrizionali di tre linee di Arabidopsis trasformate con il gene ENOD40 sono stati acquisiti e confrontati con quelli ottenuti da piante wild type. In seguito, l'analisi dei biomarcatori dei dati ottenuti dalle analisi di metabolomica e trascrittomica è stata utilizzata per identificare i metaboliti e i trascritti che hanno mostrato un maggiore correlazione con l'overespressione del gene.
Dai profili metabolici è emerso che le tre linee trasformate sono caratterizzate dalla presenza di glucosinolati, mentre i flavonoidi caratterizzano principalmente le piante wild type. Per quanto riguarda i profili trascrizionali, la maggior parte dei geni indotti nelle tre linee trasformate (12 su 23), sono correlati con processi che avvengono nella parete cellulare. Dato che, la parete cellulare determina la forma delle cellule, il gene ENOD40 potrebbe essere coinvolto in un processo che controlla la composizione e le dinamiche della parete.
Precedenti studi morfologici condotti sulle stesse linee trasformate di Arabidopsis hanno dimostrato che queste piante presentano organi con dimensioni normali ma formati da celle più piccole; inoltre protoplasti di Arabidopsis trasfettati con il gene ENOD40 sono caratterizzati da una ridotta espansione. Questi dati hanno suggerito che il gene potrebbe avere un ruolo nel mantenere le cellule in uno stadio giovane e poco differenziato.
L'osservazione che le linee trasformate di Arabidopsis accumulino glucosinolati, metabolici tipici di tessuti giovani, suggerisce che, anche dal punto di vista metabolico, le cellule trasformate hanno caratteristiche tipiche di cellule più giovani, mentre le cellule wild type accumulano maggiormente i flavonoidi, metaboliti secondari tipici dello stato differenziato.
Per quanto riguarda l'analisi trascrizionale, dal momento che le piante trasformate sono morfologicamente caratterizzate da cellule con dimensioni ridotte, i geni indotti in queste linee, potrebbero essere coinvolti nella prevenzione dell’espansione cellulare.
Questo ruolo del gene, atto a mantenere le cellule in uno stadio giovanile, è supportato anche dai profili di espressione del gene riportati in letteratura.ENOD40 is an Early Nodulin gene that it is know to play a key role in nodule formation in response to interaction of legume plants with symbiotic Rhizobium bacteria.
Homologues of ENOD40 genes have been identified in several plant species and its expression is observed during the initiation and development of new organs, such as nodules, lateral roots, young leaves and stipule primordia.
ENOD40 gene has an unusual structure: it lacks a long open reading frame, but several short ORFs are present. Moreover, at nucleotide level, two regions, named box1 and box2, are highly conserved among all ENOD40 genes. In box 1 region, a highly conserved ORF (ORF 1) is present and it seems to encode a putative peptide of 10-13 amino acids. Furthermore, the gene contains regions corresponding to conserved secondary structures of the transcript. Six domains were identified in ENOD40 mRNA and two of these domains are strongly conserved among legume and non legume species.
Despite several researches, the roles of the ENOD40 gene has not been so far completely elucidated. Moreover, whether the biological activity should be ascribed to RNA or peptide, or both, is still unclear. For this reason, the two main goals of the research are: to investigate the possible presence of the putative peptide encoded by box1 of the ENOD40 gene in BY-2 cells and to investigate the role of ENOD40 gene in non legume plants, using Arabidopsis thaliana.
That ENOD40 could act, at least in part, through the peptide encode by box1 is suggested by several observations, but no one have revealed biochemically the putative peptide.
In the first part of the work a purification procedure consisting of membrane cut-off, ion exchange chromatography, solid exchange extraction, HPLC-DAD and mass spectrometry (LC-ESI-MS and MALDI-TOF) was set up to search for the putative peptide in BY-2 cells overexpressing NtENOD40 gene. Unfortunately, despite several attempts to set up the purification procedure and the different and sensitive techniques used for the analysis of the putatively peptide-enriched fractions, only MALDI-TOF PSD analysis gave an initial clue of the possible presence of the peptide in ENOD40 overexpressing BY2 cells.
In the second part of the work, the possible role of the gene has been investigated through the metabolomics and transcriptomics characterization of ENOD40 overexpressing Arabidopsis plants.
Metabolite and transcriptional profiles of the three Arabidopsis lines overexpressing soybean ENOD40 gene were acquired and compared to those obtained from wild type plants. Afterward, biomarker analysis of metabolomic and transcriptomic dataset was used in order to identify the metabolites and transcripts that showed the higher correlation with the overexpression of ENOD40 gene.
In the metabolite profiles, glucosinolate metabolites characterized all the three transformed lines compared with the wild type, while flavonoids mainly characterized wild type plants.
With regard to transcriptional profiling, most of the genes upregulated in the three transformed lines (twelve out of twenty-three), were correlated with processes occurring in the cell wall. Thus, the cell wall is the mechanical determinant of cell shape and size ENOD40 gene could be involved in a process that controls the composition and the dynamics of the cell wall.
In conclusion, previous morphological studies on the same Arabidopsis thaliana ENOD40 transformed lines used in this work have been showed that these plants are characterised by normal organs containing smaller cells, and on ENOD40 transfected Arabidopsis protoplasts are characterized by reduced expansion, suggested that the gene could have some role in keeping the cells in a “young” state .
The observation that ENOD40 transformed Arabidopsis lines accumulate high levels of glucosinolates, that are typical of the young tissues, suggests that, also from the metabolic point of view, the transformed cells have features typical of younger cells, whereas wild type cells use their metabolic resources to accumulate flavonoids, another class of secondary metabolites more typical of differentiated state.
With regard to transcriptomic analysis, since transformed plants are morphologically characterized by small cell size, the genes upregulated in the transformed lines, involved in cell wall dynamics and composition, could be involved in the prevention of cell expansion.
The role of ENOD40 in maintenance of cells in a “young state” is also supported by the expression patterns of ENOD40 genes reported in literature
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