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Title: Involvement of polyamine metabolism in xylem differentiation and stress responses
Other Titles: Coinvolgimento del metabolismo delle poliamine nel differenziamento dello xilema e nelle risposte agli stress
Authors: Tisi, Alessandra
metadata.dc.contributor.advisor: Angelini, Riccardo
Issue Date: 19-Dec-2011
Publisher: Università degli studi Roma Tre
Abstract: Amine oxidases (AOs) include the copper-containing amine oxidases (CuAOs) and the FAD-dependent amine oxidases (PAOs). AOs oxidize polyamines (PAs) to aldehydes, producing the removed amine moiety and hydrogen peroxide (H2O2). CuAOs and PAOs contribute to important physiological processes not only through the regulation of cellular PA levels, but do so also through their reaction products. Notably, H2O2 is a product common to all AO-catalyzed reactions. H2O2 derived from PA catabolism is involved in important processes, such as plant growth and development as well as biotic and abiotic stress responses. In the present work, the involvement of polyamine metabolism in xylem differentiation and defence responses, such as wound healing, has been studied. For this purpose different plant species have been analysed, i.e. Zea mays for its agronomical importance and the well characterized apoplastic PAO (ZmPAO) present in this plant, Nicotiana tabacum and Arabidopsis thaliana for the easiness of transformation and the availability of specific mutants. In detail I took advantage of the availability of transgenic tobacco plants over-expressing ZmPAO in the apoplast (S-ZmPAO plants) or downregulating endogenous PAO (A-ZmPAO plants) or S-adenosyl-Lmethionine decarboxylase, enzyme involved in PA biosynthesis (RNAiSAMDC plants), Arabidopsis insertional mutant plants for a gene encoding for an apoplastic CuAO (Atcuao1 plants), as well as transgenic tobacco and Arabidopsis plants expressing a fungal polygalacturonase (PG plants). In maize primary root ZmPAO is mainly expressed in early metaxylem (EMX), late metaxylem (LMX), xylem parenchyma and sloughed root cap cells. In the present work it was demonstrated that the treatment of maize seedlings with the PAO substrate spermidine (Spd) induced the deposition of cell wall phenolics in vascular tissues and xylem parenchyma of primary root, likely associated to early maturation of the cell wall. Importantly, this effect correlated with a high production of H2O2 in the same tissues upon Spd treatment. Consistently, in presence of exogenous Spd, immature LMX and EMX cells appeared closer to the apical meristem than in Spd-untreated plants. The early differentiation of xylem cells associated with enhanced H2O2 production in Spd-treated roots suggests the hypothesis that vascular tissue differentiation could be mediated by the PAO-driven Spd oxidation. Further evidence in support of this hypothesis came from the analysis of SZmPAO tobacco plants. In these plants PAO over-expression in the cell wall resulted in early differentiation of root tracheary elements and cell death of sloughed root cap cells and rhizodermis of the subapical region, as well as enhanced in vivo H2O2 production in xylem tissues. Unexpectedly, vascular differentiation in A-ZmPAO tobacco plants occurred in a similar fashion as compared to wild type (WT) plants. Remarkably, downregulation of SAMDC in tobacco promoted xylem cell differentiation and induced PCD in root cap cells similarly to ZmPAO over-expression in the cell wall, suggesting that xylem differentiation and PCD induction are governed by a complex regulatory system in which intracellular PAs and apoplastic PA-derived H2O2 are likely integrated in coordinated signaling pathways. In Arabidopsis a gene encoding for an apoplastic CuAO, expressed in root vascular tissue at early developmental stage was already identified (AtCuAO1 gene). In this work it was demonstrated that AtCuAO1 expression was induced by methyl jasmonate (MeJA) treatment but not wounding. Interestingly, MeJA is a plant hormone involved in defence responses and xylem development. In normal physiological conditions, Atcuao1 mutant plants did not show defective phenotype in comparison to WT plants. MeJA treatment resulted in early xylem differentiation in Arabidopsis WT seedlings, without affecting xylem differentiation in Atcuao1 mutant seedlings. Consistently, the analysis of polyamine levels showed that putrescine (Put) level was selectively impaired upon MeJA supply in WT roots but it did not change in mutant roots. The reversion effect exerted by the H2O2 scavenger DMTU on MeJA-induced xylem differentiation in WT roots along with the histochemically revealed production of H2O2 at the site of differentiating xylem upon MeJA treatment in WT roots, provided evidence for the specific involvement of the AtCuAO1-produced H2O2 in root xylem differentiation. Furthermore, treatment with Put, AtCuAO1 substrate, induced early xylem differentiation in WT but not in Atcuao1 roots, effect reversible by DMTU treatment, suggesting an indirect effect of Put mediated by H2O2. Since the existence of signal crosstalk between MeJA and abscissic acid (ABA) has been suggested to occur during drought conditions, the effect of ABA treatment on the xylem differentiation of WT and Atcuao1 plants was analyzed. However, unlike MeJA, ABA equally affected WT and Atcuao1 roots, inducing early xylem differentiation to the same extent in both WT and mutant plants. The possible involvement of AO in wound healing and xylem differentiation was also studied in transgenic tobacco plants expressing a fungal polygalacturonase (PG tobacco plants) that show enhanced defence responses, associated with accumulation of reactive oxygen species and constitutive expression of several defence genes. Present results obtained with PG tobacco plants suggest that H2O2 derived from PA catabolism may behave as a mediator in the signalling pathway triggered by oligogalacturonides (OG), compounds derived from partial degradation of the cell wall component homogalacturonan by fungal polygalacturonases. In particular, higher CuAO activity and lower polyamine levels, particularly Put, were found in shoot tissues of PG tobacco plants. In accord with the enhanced defence response peculiar of PG plants, wound responses appeared to be improved in transgenic plants as well, which exhibited an intensification of autofluorescence upon wounding not only in wound surface, as observed in WT plants, but also in epidermis and cortical parenchyma. Using a pharmacological approach, the involvement of CuAO enzyme activity in the distinctive behaviour shown by PG plants upon wounding, characterized by the spreading of defence signals also in wound adjacent tissues, has been suggested. In agreement with the stronger CuAO activity revealed by DAB staining in xylem cells of PG plants in comparison to xylem tissues of WT plants, early xylem differentiation was observed in PG tobacco roots. This phenotype could be reasonably ascribed to a higher level of CuAO activity in PG plants as compared to WT plants because of the lower level of Put detected in root of PG plants in comparison to WT root as well as the reversion effect of the CuAO inhibitor 2-bromoethylamine on xylem differentiation and H2O2 accumulation level in roots of PG plants. Analogously to tobacco PG plants, roots of Arabidopsis PG plants displayed Put depletion (Dr. Sandip A. Ghuge, Dept. of Biology, University “Roma Tre”, personal communication) and early xylem differentiation associated to enhanced in situ apoplastic H2O2 production. Overall, the lack of a defective phenotype showed by both transgenic tobacco plants down-regulating endogenous PAO and untreated Arabidopsis Atcuao1 plants apparently contrasts with the hypothesis that PA catabolism represents a key element in xylem differentiation during development under normal physiological conditions. However, this incongruity could be explained bearing in mind that the observed early xylem differentiation associable to AO activity was exclusively revealed in plants undergone to stress-like conditions linked to enhanced extracellular H2O2 production and/or imbalanced PA/H2O2 ratio, obtained by both pharmacological and genetic approaches able to alter PA levels. Hence, we can hypothesize that H2O2 derived from PA catabolism has a role in inducing xylem differentiation under stress conditions, such as those simulated by increased PA oxidation in the cell wall, while further investigations are needed to establish its role under normal physiological conditions.
Access Rights: info:eu-repo/semantics/openAccess
Appears in Collections:X_Dipartimento di Biologia
T - Tesi di dottorato

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