Konstantinos A. Paschalidis

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the α-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the α-subunit of GDH, increase in immunoreactive α-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy–mass spectrometry monitoring of 15N-Glu, 15N-Gln, and 15N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of α-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive α-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces α-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

Spatial and temporal distribution of polyamine levels and polyamine anabolism in different organs/tissues of the tobacco plant. Correlations with age, cell division/expansion, and differentiation.

Polyamine (PA) titers and biosynthesis follow a basipetal decrease along the tobacco (Nicotiana tabacum) plant axis, and they also correlate negatively with cell size. On the contrary, the titers of arginine (Arg), ornithine (Orn), and arginase activity increase with age. The free (soluble)/total-PA ratios gradually increase basipetally, but the soluble conjugated decrease, with spermidine (Spd) mainly to determine these changes. The shoot apical meristems are the main site of Spd and spermine biosynthesis, and the hypogeous tissues synthesize mostly putrescine (Put). High and low Spd syntheses are correlated with cell division and expansion, respectively. Put biosynthetic pathways are differently regulated in hyper- and hypogeous tobacco tissues: Only Arg decarboxylase is responsible for Put synthesis in old hypergeous vascular tissues, whereas, in hypogeous tissues, arginase-catalyzed Orn produces Put via Orn decarboxylase. Furthermore, Orn decarboxylase expression coincides with early cell divisions in marginal sectors of the lamina, and Spd synthase strongly correlates with later cell divisions in the vascular regions. This detailed spatial and temporal profile of the free, soluble-conjugated, and insoluble-conjugated fractions of Put, Spd, and spermine in nearly all tobacco plant organs and the profile of enzymes of PA biosynthesis at the transcript, protein, and specific activity levels, along with the endogenous concentrations of the precursor amino acids Arg and Orn, offer new insight for further understanding the physiological role(s) of PAs. The results are discussed in the light of age dependence, cell division/expansion, differentiation, phytohormone gradients, senescence, and sink-source relationships.

Plant polyamine catabolism: The state of the art

Polyamines have long been implicated in plant adaptation to abiotic and biotic stress. As a general rule of thumb the higher the polyamine titers the better. However, their molecular roles in plant stress responses still remain obscure. It has been postulated that they could act through their catabolism, which generates molecules which may act as secondary messengers signalling networks of numerous developmental and stress adaptation processes. Recently it was shown that plant and mammalian polyamine catabolism share critical features, giving new insight in plant polyamine catabolism. In this review, the advances in genes and proteins of polyamine catabolism in plants is presented and compared to other models.

Sites and Regulation of Polyamine Catabolism in the Tobacco Plant. Correlations with Cell Division/Expansion, Cell Cycle Progression, and Vascular Development

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development.

Abscisic acid signals reorientation of polyamine metabolism to orchestrate stress responses via the polyamine exodus pathway in grapevine.

Polyamines (PAs) have been suggested to be implicated in plant responses to abiotic and biotic stress. Grapevine is a model perennial plant species whose cultivars respond differently to osmotic stress. In this study, we used two cultivars, one sensitive (S) and one tolerant (T) to drought. In adult vines subjected to drought under greenhouse conditions, total PAs were significantly lower in the control T- and higher in the control S-genotype and significantly increased or decreased, respectively, post-treatment. Soluble Put and Spd exhibited the greatest increase on d 8 post-treatment in the T- but not in the S-genotype, which accumulated soluble Spm. Abscisic acid (ABA) was differentially accumulated in T- and S-genotypes under drought conditions, and activated the PA biosynthetic pathway, which in turn was correlated with the differential increases in PA titers. In parallel, polyamine oxidases (PAOs) increased primarily in the S-genotype. ABA at least partially induced PA accumulation and exodus into the apoplast, where they were oxidized by the apoplastic amine oxidases (AOs), producing H2O2, which signaled secondary stress responses. The results here show that the ABA signaling pathway integrates PAs and AOs to regulate the generation of H2O2, which signals further stress responses or the PCD syndrome.

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors.

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.

Engineered Polyamine Catabolism Preinduces Tolerance of Tobacco to Bacteria and Oomycetes

Polyamine oxidase (PAO) catalyzes the oxidative catabolism of spermidine and spermine, generating hydrogen peroxide. In wild-type tobacco (Nicotiana tabacum ‘Xanthi’) plants, infection by the compatible pathogen Pseudomonas syringae pv tabaci resulted in increased PAO gene and corresponding PAO enzyme activities; polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine was excreted into the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher hydrogen peroxide accumulation. Moreover, plants overexpressing PAO showed preinduced disease tolerance against the biotrophic bacterium P. syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in a specific apoplast-localized plant-pathogen interaction, which also signals parallel defense responses, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of plant pathogens.

Polyamine anabolic/catabolic regulation along the woody grapevine plant axis ☆

The distribution of the endogenous PA fractions throughout the entire perennial woody grapevine (Vitis vinifera L.) plant was studied, along with the expression profiles of the PA anabolic and catabolic genes and their substrates and secondary metabolites. Putrescine fractions increased with increasing leaf age, although the expression of its biosynthetic enzymes Arg and Orn decarboxylases decreased. Orn transport from young organs dramatically enhanced putrescine biosynthesis in older tissues, via the Orn decarboxylase pathway. S-adenosylmethionine decarboxylase and spermidine synthase genes were down-regulated during development in a tissue/organ-specific manner, as were spermidine and spermine levels. In contrast, amine oxidases, peroxidases and phenolics increased from the youngest to the fully developed vascular tissues; they also increased from the peripheral regions of leaves to the petioles. Hydrogen peroxide generated by amine oxidases accumulated for the covalent linkage of proteins via peroxidases during lignification. These results could be valuable for addressing further questions on the role of PAs in plant development.

POLYAMINES IN GRAPEVINE: An Update

The polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) exist as free (soluble; S-), or as conjugated to small (SH-) or to large (PH-) molecules. Increasing evidence supports that PAs represent important in-trinsic developmental signals, which may affect developmental processes that range from cell division and morphogenesis to stress responses (Perez-Amador et al. 2002, Fos et al. 2003, Arias et al. 2005, Paschalidis et al. 2001, Paschalidis and Roubelakis-Angelakis 2005a, b, Cona et al. 2006, Vuosku et al. 2006, Ku-sano et al. 2007, Agalou et al. 2008, Moschou et al. 2008a, b, c). The first step in PA biosynthesis is the formation of Put from Orn and Arg