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The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.

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Polyamines (PAs), such as spermidine and spermine, as well as amino acids that are substrates for their biosynthesis, are known to be essential for plant development. However, little is known about the gene expression and metabolic switches associated with the ornithine/arginine and PA biosynthetic pathway during seed development in conifers. To understand these metabolic switches, the enzyme activity of arginine decarboxylase and ornithine decarboxylase, as well as the contents of PAs and amino acids were evaluated in three Araucaria angustifolia (Bertol. Kuntze) seed developmental stages in combination with expression profile analyses of genes associated with the ornithine/arginine and PA biosynthetic pathway. Twelve genes were selected for further analysis and it was shown that the expression profiles of AaADC and AaSAMDC were up-regulated during zygotic embryo development. Polyamines and amino acids were found to accumulate differently in embryos and megagametophytes, and the transition from the globular to the cotyledonary stage was marked by an increase in free and conjugated spermidine and spermine contents. Putrescine is made from arginine, which was present at low content at the late embryogenesis stage, when high content of citrulline was observed. Differences in amino acids, PAs and gene expression profiles of biosynthetic genes at specific seed stages and at each seed transition stage were investigated, providing insights into molecular and physiological aspects of conifer embryogenesis for use in future both basic and applied studies.

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Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia.

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

to be linked to the parasite load in lesion sites 3. In MCL lesions, parasites are rarely detected whereas in DCL lesions heavily parasitized macrophages are usually observed 2. We have previously shown high concentrations of arginase-1 (ARG1), ornithine decarboxylase (ODC), prostaglandin E2 (PGE2) and transforming growth factor β (TGF-β) in DCL patients 4 , which could contribute to an ineffective immune response unable to hamper parasite replication. Although recent studies have shown that components of the polyamine biosynthetic pathway are linked to survival of Leishmania sp. inside macrophages in experimental settings 5,6 it is unknown whether there is a differential expression of such components in patients with distinct clinical forms of TL. Among the metabolites from the polyamine pathway, putrescine, cadaverin, spermidine and spermine are aliphatic cations derived from amino acids such as l-arginine and lysine, with multiple functions which are essential for all living organisms 7. Polyamines are critically involved in a diverse range of cellular processes such as regulation of gene expression and translation, modulation of cell signaling, membrane stabilization and cell proliferation 7,8. These metabolites are synthesized in a reaction catalyzed by ARG1, which converts l-arginine to l-ornithine and urea 6. Another enzyme, ODC, catalyzes l-ornithine conversion to putrescine 6. Putrescine then participates in an intricate cascade of reactions involving several enzymes such as spermidine synthase (SpdS) and spermine synthase (SpmS), which results in formation of polyamines, spermidine and spermine, respectively 6. Cadaverine, a polyamine poorly studied in humans, is derived from the amino acid lysine 9. The uptake of l-arginine in macrophages infected with Leishmania sp. occurs via transporters from the cationic amino acid family (CAT) 10. Hence, inhibition of the l-arginine transporter by melatonin reduces parasite burden by decreasing the production of polyamines 11. We have previously demonstrated that treatment of L. amazonensis infected macrophages with arginase or ODC inhibitors leads to enhanced parasite clearance and dampened secretion of pro-inflammatory cytokines 4. Indeed, different immune response profiles can influence l-arginine catabolism that, ultimately, result in resistance or susceptibility to Leishmania infection. l-arginine is catabolized by ARG1 in the presence of interleukin 4 (IL-4), IL-10, IL-13 and TGF-β, producing polyamines and collagen and enhancing Leishmania infection 12. In converse, in the presence of pro-inflammatory mediators, such as interferon γ (IFNγ), tumor necrosis factor α (TNFα) and IL-12, the nitric oxide synthase 2 (iNOS/NOS2) will be preferentially activated, resulting in production of nitric oxide (NO) and citrulline 12,13. Although NO alone is not sufficient to control infection, it can be further metabolized in reactive nitrogen and oxygen species, which are then involved in parasite killing 14,15. Therefore, the profile of the host immune responses dictates ...