Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

Targeted gene disruption coupled with metabolic screen approach to uncover the LEAFY COTYLEDON1-LIKE4 (L1L4) function in tomato fruit metabolism.

KEY MESSAGE: Functional analysis of tomato L1L4 master transcription factor resulted in important metabolic changes affecting tomato fruit quality. Tomato fruits from mutant lines bearing targeted disruption of the heterotrimeric nuclear transcription factor Y (NF-Y) transcription factor (TF) gene LEAFY-COTYLEDON1-LIKE4 (L1L4, NF-YB6), a master regulator of biosynthesis for seed storage proteins and fatty acids, were evaluated for metabolites content and morphology. Metabolic screens using LC-MS/MS-based analysis and physico-chemical methods in different L1L4 mutants of the fourth generation allowed a comparative assessment of the effects of the TF disruption. Mutagenesis resulted in fruits phenotypically similar to wild-type with subtle shape differences in the distal end protrusion and symmetry. Conversely, mutant fruits from independent lines had significant variation in moisture content, titratable acidity and overall metabolite profiles including oxalic and citric acid, fructose, ß-carotene, total polyphenols and antioxidants. Lines 6, 7 and 9 were the richest in ß-carotene and antioxidant activity, line 4 in ascorbic acid and lines 4 and 8 in succinic acid. The reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene may regulate the accumulation of this compound during fruit development. Detailed LC-MS/MS analysis of mutant seeds showed substantial differences in bioactive compounds compared to wild-type seeds. Taken together, the results suggest that the L1L4 TF is a significant regulator of metabolites both in tomato fruit and seeds providing a molecular target for crop improvement. Elucidation of the candidate genes encoding key enzymes in the affected metabolic pathways aimed to facilitate the L1L4 gene network exploration and eventually lead to systems biology approaches in tomato fruit quality.

Mixed epithelial and stromal tumor of the kidney (MEST) simulating an upper tract TCC.

We present a rare and interesting case of a mixed epithelial and stromal tumour (MEST) of the kidney. The case is unique as it involves a male patient with no history of hormonal therapy presenting with a filling defect in the renal collecting system and positive urine cytology. The patient was diagnosed with transitional cell carcinoma of the renal pelvis and subjected to nephroureterectomy, which revealed a solid tumour arising from the lower calyces and extending into the renal pelvis and upper ureter. Pathology revealed a MEST. The patient was disease-free at the 6-month follow-up.

Secondary malignancies following radiotherapy for prostate cancer.

Human exposure to sources of radiation as well as the use of radiation-derived therapeutic and diagnostic modalities for medical reasons has been ongoing for the last 60 years or so. The carcinogenetic effect of radiation either due to accidental exposure or use of radiation for the treatment of cancer has been undoubtedly proven during the last decades. The role of radiation therapy in the treatment of patients with prostate cancer is constantly increasing as less-invasive treatment modalities are sought for the management of this widely, prevalent disease. Moreover the wide adoption of screening for prostate cancer has led to a decrease in the average age that patients are diagnosed with prostate cancer. Screening has also resulted in the diagnosis of low-grade, less-aggressive prostate cancers which would probably never lead to complications or death from the disease. Radiotherapy for prostate cancer has been linked to the late occurrence of second malignancies both in the true pelvis and outside the targeted area due to low-dose radiation scatter. Secondary malignancies following prostate irradiation include predominantly bladder cancer and, to a lesser extent, colon cancer. Those secondary radiation-induced bladder tumors are usually aggressive and sometimes lethal. Care should be given to the long-term follow up of patients under radiation therapy for prostate cancer, while the indications for its use in certain cases should be reconsidered.

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.

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.

Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco.

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.

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.

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 alpha-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 alpha-subunit of GDH, increase in immunoreactive alpha-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 (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-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 alpha-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 alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

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.

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.