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Title: Advances in the involvement of polyamine oxidases in Arabidopsis thaliana cellular metabolism
Authors: Mancuso, Namrata
Advisor: TAVLADORAKI, Paraskevi
Issue Date: 1-Mar-2018
Publisher: Università degli studi Roma Tre
Abstract: In plants, the polyamines putrescine, spermidine (Spd), spermine (Spm) and thermospermine (Therm-Spm) are involved in the regulation of several cellular processes, such cell proliferation and differentiation, programmed cell-death, and defense responses. In particular, Therm-Spm, a structural isomer of Spm, is important for normal growth and development, since it is involved in the control of xylem differentiation having an auxin antagonizing effect. In Arabidopsis thaliana, five polyamines oxidases (AtPAO1–AtPAO5) are present involved in polyamine catabolism. AtPAO1 and AtPAO5 are cytosolic enzymes catalyzing the back-conversion of Spm and Therm-Spm to Spd. AtPAO5 is also able to oxidize N1-acetyl-Spm. Conversely, the other three members of the Arabidopsis PAO family, AtPAO2, AtPAO3 and AtPAO4, have a peroxisomal localization and are able to oxidize both Spd and Spm, but not Therm-Spm. Furthermore, based on the high sequence homology and the similar gene structure the three peroxisomal AtPAOs are considered recent derivatives of a common ancestor gene and thus to form a distinct subfamily. Studies on the tissue- and organ-specific expression pattern of the five AtPAOs using AtPAO::GFP-GUS Arabidopsis transgenic plants, showed common, but also distinct expression patterns for each one of the five AtPAOs. Characteristic expression patterns of AtPAO1 is the transition region between the meristematic and the elongation zone of the root and the anther tapetum, of AtPAO2, AtPAO3 and AtPAO4 the guard cells and pollen grains, while of AtPAO5 the vascular tissue and the anther tapetum. In the present study, quantitative Real-time RT-PCR (qRT-PCR) analysis evidenced that AtPAO1, AtPAO2 and AtPAO3 expression levels are modulated by the stress related hormones abscisic acid (ABA) and methyl Jasmonate (MeJA), as well as by stress conditions (NaCl treatment and drought), while on the contrary, the expression levels of AtPAO4 and AtPAO5 remain invariable under these conditions. Recent studies have shown that atpao2, atpao3 and atpao4 insertional mutants display reduced stomatal closure, as compared to wild-type plants, in response to ABA. Interestingly, the reduced stomata closure observed in the single mutants was even more pronounced in the double atpao2atpao4 and atpao3atpao4 mutants and the triple atpao2atpao3atpao4 (atpao234) mutants, the last one presenting the highest variation in stomata movement in respect to the wild-type plants. This suggests the involvement of all three peroxisomal AtPAOs in the ABA-mediated control of guard cells in a synergistic way. Here, further studies were performed using the atpo234 triple mutant and AtPAO3 over-expressing Arabidopsis transgenic plants (AtPAO3 transgenic plants) to better understand the contribution of the AtPAO234 gene subfamily in the control of stomata movement. It was shown that atpao234 triple mutant, but not the AtPAO3 transgenic plants, displays reduced stomata closure not only in response to ABA but also to MeJA, H2O2 and polyamines, as compared the wild-type plants. The underlying mechanisms in the reduced stomata closure exhibited by the atpao234 plants are not clear so far. However, reduced production of H2O2, which is an important second messenger in the ABA signaling network, as well as variations in polyamine levels, which play an important role in the modulation stomata movement, probably do not contribute to this effect. Noteworthy, the reduced responsiveness of the atpao234 triple mutant to the stress-related stimuli leading to stomata closure, which should contribute to reduce water loss through transpiration is not accompanied by increased tolerance to abiotic stress conditions. The lack of such a correlation has still to be analyzed. It was recently shown that AtPAO5 is involved in the control of plant growth. Indeed, two atpao5 mutants and an AtPAO5 over-expressing Arabidopsis transgenic lines (AtPAO5 transgenic plants) present developmental differences from the wild-type plants. In particular, the atpao5 mutants produce longer and thicker flowering stems, while the AtPAO5 plants produce thinner and shorter stems as compared to the wild-type plants. These phenotypical alterations of atpao5 mutants and AtPAO5 transgenic plants were attributed to AtPAO5-mediated changes in Therm-Spm homeostasis and are accompanied by changes in the expression level of genes involved in auxin and cytokinin signaling, suggesting that AtPAO5 interferes with cytokinin and auxin signaling pathways. In the present study, to further investigate on the auxin signaling in the AtPAO5 and atpao5 plants, sexual crossings of these plants with DR5::GUS transgenic plants, DR5 being an artificial auxin–regulated promoter, were performed and analyzed. It was shown that the AtPAO5 present increased DR5::GUS expression levels in the root meristematic region, whereas atpao5 decreased as compared the DR5::GUS expression levels in wild-type roots. It was further shown that the AtPAO5 and atpao5 plants present differences from wild-type plants regarding xylem differentiation throughout the entire plant, with AtPAO5 plants presenting an increased number of xylem vessel elements and atpao5 mutants a decreased number compared with wild-type plants. Furthermore, it was shown that the atpao5 plants exhibit increased tolerance to salt stress and drought, as well as decreased stomata closure in response to stress-related conditions. In conclusion, the presented data evidence an important role of the three peroxisomal AtPAOs in the control of stress-mediated stomata closure. It was also shown a tightly controlled interplay between AtPAO5, Therm-Spm, auxin and cytokinins necessary for proper xylem differentiation and plant growth. AtPAO5 most probably contributes to this regulatory network participating in the feedback mechanisms which control Therm-Spm levels. On the other hand, AtPAO5 participates in the control of stomata closure and plant tolerance to salt and drought stress.
Access Rights: info:eu-repo/semantics/openAccess
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