Search

Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of ∼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.

An improved understanding of seed quality and germination control can contribute effectively to the use and conservation of neglected native species with ecological and economic value, such as Trichocline catharinensis, an endemic Asteraceae species from southern Brazil. We investigated the effects of applying gibberellin (GA3), abscisic acid (ABA) and their biosynthesis inhibitors, paclobutrazol (PAC) and fluridone (FLU), respectively, on T. catharinensis seed germination, and on polyamine (PA) content and antioxidant enzyme activities in germinating seeds. FLU and GA3 increased seed germination speed compared to treatment with H2O only. ABA inhibited both germination speed index and percentage, while PAC severely inhibited seed germination. The stimulatory effect of GA3 and FLU was associated with increased contents of putrescine (PUT) and spermidine (SPD) relative to spermine (SPM). As a result, high ratio (PUT + SPD/SPM) as well as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) enzyme activities were observed when seed germination occurs. In contrast, in low or no seed germination treatment (ABA and PAC), low ratio (PUT + SPD/SPM) was observed, while the antioxidant enzymes, mainly SOD activity, tended to increase. Application of PAs at 200 μM stimulated germination through improving the speed and uniformity, and this effect was associated with antioxidant enzyme activity. Our results suggest a relationship between PA and the antioxidant system with the physiological mechanism of seed germination. These results improve the physiological knowledge of seed germination control in Asteraceae and contribute to the biological groundwork for future studies on the use and conservation of native species.

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was signifi- cant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0 mM and GSH 0.1 mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5 mM treatment showed constantlevels. Alltreatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5 mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized

Guadua chacoensis (Poaceae) is a woody bamboo native from the Atlantic forest biome. Morphogenetic and physiological studies are scarce in bamboos, and tissue culture-based biotechnologies tools can be used to investigate ultrastructure and physiological processes as well as to mass-propagate specific genotypes. This study evaluated the effect of light and dark conditions on chloroplast biogenesis as well as in the endogenous levels of zeatin (Z), abscisic acid (ABA), gibberellic acid (GA4), and jasmonic acid (JA) during in vitro culture of G. chacoensis. An increase was observed, followed by a decrease in starch content in response to light treatment, and in contrast, in darkness, an accumulation of starch which is associated to amyloplast formation at day 30 was observed. No etioplast formation was observed even in the dark and this was associated with the presence of fully developed chloroplast at the beginning of the experiment. Z levels quantified showed distinct behavior, as in light, no difference in the levels was observed, except at day 10, and in darkness, the levels increased along the evaluation time. ABA, GA4, and JA biosynthesis increased along the time of evaluation in light condition. In contrast, in darkness, the levels remained unaltered, excepted for an increase in JA levels at day 10, suggesting a strong relationship between light and these phytohormone levels. Possible reasons why light’s presence and absence cause changes in chloroplast ultrastructure and hormone endogenous levels in in vitro cultivated culms of G. chacoensis are discussed. Keywords Bamboos · Micropropagation · Transmission electron microscopy · Plant physiology · Phytohormone Made available in DSpace on 2019-03-02T00:37:13Z (GMT). No. of bitstreams: 1 Polesi2019ArticleChloroplastUltrastructureAndHo.pdf: 1356355 bytes, checksum: 25b47e78e8251e1ff16794227f651cc5 (MD5) Previous issue date: 2019-01-10

Butia eriospatha is an endemic palm species from the Atlantic Rainforest in Brazil, a biodiversity hotspot. This species is currently listed in the IUCN red list as vulnerable and lacks specific plastid markers for population genetics studies. In addition, the evolutionary relationship within the genus Butia is not yet well resolved. Here, we sequenced and characterized the complete plastid genome (plastome) sequence of B. eriospatha. The complete plastome sequence is 154,048 bp in length, with the typical quadripartite structure. This plastome length and genes content is consistent with other six species from tribe Cocoseae. However, the Inverted Repeat (IR) borders show some variation among the analyzed species from this tribe. Species from the Bactridinae (Astrocaryum and Acrocomia) and Elaeidinae (Elaeis) subtribes present the rps19 gene completely duplicated in the IR region. In contrast, all plastomes sequenced from the subtribe Attaleinae (Butia, Cocos, Syagrus) present one complete CDS of rps19 and one partial copy of rps19. The difference in the IR/LSC junctions between Attaleinae and the sister clades Bactridinae + Elaeidinae might be considered an evolutionary signal and the plastome sequence of B. eriopatha may be used in future studies of population genetics and phylogeny.

18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0, Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation., This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT (“Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301“), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, “Investing in your future”). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017).

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23ºC. Cultures initiated at moderate temperatures (30ºC, 4 weeks and 40ºC, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50ºC, 5 minutes). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40ºC 4 hours; 50ºC, 30 minutes; and a pulse treatment of 60ºC, 5 minutes. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites. Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23◦C. Cultures initiated at moderate temperatures (30◦C, 4 weeks and 40◦C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50◦C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40◦C 4 h; 50◦C, 30 min; and a pulse treatment of 60◦C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

Tuberculosis affects about 100 million people worldwide and causes nearly 2 million deaths annually. It has been estimated that one third of all humans is infected with latent Mycobacterium tuberculosis (Mtb). Moreover, Mtb has become increasingly resistant to available antibiotics. Consequently, it is important to identify and characterize new therapeutic targets in Mtb and to synthesize selective inhibitors. ClpP1, ClpP2 and their associated regulatory ATPases, ClpX and ClpC1 are required for the growth of Mtb and for its virulence during murine infection and are highly attractive drug targets, especially since they are not present in the cytosol of mammalian cells, and they differ markedly from the mitochondrial ClpP complex. The importance of these proteins in Mtb is emphasized by the existence of several natural antibiotics targeting this system. In order to find new inhibitors of ClpC1P1P2 system, we developed an assay based on the ATP-dependent degradation of a fluorescent protein substrate. The hits obtained were further characterized with a set of secondary assays to identify precise targets within a complex. A large library of compounds was screened and led to the identification of a ClpC1 ATPase inhibitor demonstrating that this approach can be used in future searches for anti-TB agents.

Araucaria angustifolia is a critically endangered conifer native to South America, and somatic embryogenesis (SE) is one of the most promising biotechnological tools for its conservation and mass propagation. In vitro tissue culture and chemical compounds supplemented to culture medium, especially plant growth regulators (PGRs), are known to affect DNA methylation and protein expression profiles, modulating the phenotype and/or the embryogenic potential. Here, we evaluated the global DNA methylation (GDM) levels of A. angustifolia embryogenic cultures (EC) during SE induction and multiplication steps for 1 year subcultures, and identified a wide range of differentially expressed proteins in PGR-free or -supplemented treatments. During long-term subcultures, PGR-supplementation proved to gradually increase the GDM, which may compromise genomic stability and evoke gene expression modifications. Label-free proteomics enabled a robust protein identification and quantification in A. angustifolia EC. Exclusively expression of PIN-like protein in PGR-supplemented treatment indicated a possible differential response of the EC to polar auxin transport, which can generate implications in its morphogenetic response to maturation step. Up-regulation of stress-related proteins in EC from PGR-supplemented treatment suggests its more stressful environment, triggering notable responses to hormonal, osmotic and oxidative stresses. Improved expression of proteins involved with protein folding and stabilization processes in PGR-free treatment could play a protective function in response to stress conditions caused by in vitro culture, and may provide an adaptive advantage to these EC. The expression of several proteins associated to terpenoid biosynthesis suggests that EC from both treatments and cell lines are possibly producing these compounds.

Aleppo pine (Pinus halepensis Mill.), a native species of the Mediterranean region, has been suggested as a species that when introduced in degraded areas could facilitate the long-term colonization and expansion of late-successional species. Due to climate changes, plants need to withstand extreme environmental conditions through adaptation and changings in developmental pathways. Among other paths, plants undergo changes in developmental pathways controlled by phytohormones. At the same time, somatic embryogenesis has been widely used as a model to understand the mechanisms involved in plant response to different stresses. In this study, in order to induce a strong effect of temperature stress on plants regenerated from somatic embryos, higher temperatures (40 &deg, C for 4 h, 50 &deg, C for 30 min, and 60 &deg, C for 5 min) than the control (23 &deg, C) were applied during the induction stage of somatic embryogenesis in Pinus halepensis. A morphological characterization of the embryogenic cultures showed small differences in the number of starch grains, lipid bodies, and phenolic compounds between treatments. Results showed that high temperatures (60 &deg, C) led to higher rates at the maturation stage of somatic embryogenesis when compared to the control (23 &deg, C), strengthening the productivity through the increase in the number of somatic embryos obtained. Finally, analysis of endogenous concentration of cytokinins showed that different conditions applied during the initiation phase of somatic embryogenesis led to different hormonal profiles, isoprenoid cytokinins showed a clear defined pattern with the higher total hormone concentration being found in embryonal masses induced at 50 &deg, C for 30 min, while different aromatic cytokinins presented different individual responses to the treatments applied. These differences corroborate the idea that cytokinins could be potential regulators of stress&ndash, response processes during initial steps of somatic embryogenesis.

Somatic embryogenesis (SE) has been routinely used as mass micropropagation technique, and as a model system for investigating the structural, physiological, and molecular events occurring during somatic embryo development. Successful in vitro SE is related to the quality and yield of somatic embryos obtained. In cacao (Theobroma cacao L.) SE, the efficiency of somatic embryo production has improved with secondary SE. However, the low number of somatic embryos able to conversion into viable plantlets is still low. Then, two morphological types of normal mature somatic embryos can be identified during cacao secondary SE. The first type shows white appearance and high conversion potential (75%), while the second type shows translucent appearance and exhibit low conversion potential (15%). In order to investigate the proteins that can be associated to conversion potential in cacao somatic embryos, the mass spectrometry HDMSE proteomic approach was used. At least 60 proteins showed differences in abundance levels in cacao white somatic embryos, when compared to translucent. An increased abundance of Beta-glucosidase, NAD(P)-linked oxidoreductase and Electron transfer flavoprotein proteins were observed in white somatic embryos. Moreover, in translucent somatic embryos were observed an increased abundance of Cytochrome P450 and Pathogenesis-related proteins. Using white somatic embryos as a model, we suggest that carbohydrate metabolism process and the redox regulation are involved in the control/regulation of somatic embryo quality. These new findings may improve cacao SE protocol, as well as the understanding of the role of pivotal metabolic pathways associated to this in vitro morphogenetic route.

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

International audience; Solid-state NMR can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here we show the use of two sets of experiments that expand the tool kit of 1H-detected assignment approaches, and which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to up to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. We exemplify the usefulness of these experiments with applications to assemblies of deuterated, fully amide-protonated proteins from ca. 20 to 60 kDa monomer, at MAS frequencies from ca. 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. We report the resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6, and compare these to solution-state NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly. Solid‐state NMR H‐N‐(C)‐H and H‐N‐C‐C 3D/4D correlation experiments for resonance assignment of large proteins. Available from: https://www.researchgate.net/publication/319020315_Solid-state_NMR_H-N-C-H_and_H-N-C-C_3D4D_correlation_experiments_for_resonance_assignment_of_large_proteins.

In Theobroma cacao L., declined embryogenic potential was observed in regenerated somatic embryos from long-term secondary somatic embryogenesis (SE). In order to explore the relationship between DNA methylation and the long-term secondary SE, the embryogenic potential and global DNA methylation levels in young (12 months-old), aged (36 months-old) and extra somatic embryogenesis (39 months-old) subjected to different 5-Azacytidine (5-azaC) treatments were comparatively assessed. Global DNA methylation levels increased in aged somatic embryos with long-term in vitro culture, but 5-azaC-supplemented treatments resulted in unaltered levels. In addition, DNA methylation pattern during SE was not affected by 5-azaC. DNA methylation increased during SE expression. Interestingly, the extra SE induction showed that aged somatic embryos can recovery the embryogenic potential in treatment supplemented with 5-azaC at specific concentration. The outcome of this study suggested that the long-term SE in cacao induced the decline on embryogenic potential, which can be reversible trough 5-azaC supplementation. Besides, increased DNA methylation levels might be a response to the stress conditions that plant cells were exposed to during SE.

Somatic embryogenesis (SE) is a complex, multifactorial model of molecular, biochemical, physiological and developmental steps of plant embryogenesis. This regenerative route arises from a single totipotent somatic cell that has dedifferentiated or redifferentiated into an embryonic state. Additionally, it comprises a high performance in vitro regenerative system for both the mass clonal propagation of an elite genotype and/or the mass propagation of endangered plant germplasm. Following coordinated subsequent cell divisions, changes in cellular contents and balances of protein, hormones, polyamines, polysaccharides, and lipids, histodifferentiation processes takes place, culminating with the full development of a mature somatic embryo, mimicking the developmental stages observed in a zygotic embryo. These dynamics are simultaneously affected by a myriad of factors, mainly associated to epigenetic reprogramming, tissue culture environment, culture media composition, and type, levels and balances of plant growth regulators. In this context, in the present review we present and discuss three SE systems of selected and typical Brazilian native woody species, representing different taxonomic groups: Araucaria angustifolia (Araucariaceae), Acca sellowiana (Myrtaceae) and Bactris gasipaes (Arecaceae). Our approach to SE wants to contribute to the better understanding of plant cell totipotentiality, as well as to address its application in capturing genetic gains from elite genotypes, and for use in conservation programs of endangered species. Fil: Guerra, M.P.. Universidade Federal de Santa Catarina; Brasil Fil: Fraga, H.P.F.. Universidade Federal de Santa Catarina; Brasil Fil: Vieira, L.N.. Universidade Federal de Santa Catarina; Brasil Fil: Ree, J.F.. Universidade Federal de Santa Catarina; Brasil Fil: Heringer, A.S.. Universidade Federal de Santa Catarina; Brasil Fil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

This chapter deals with the features of somatic embryogenesis (SE) in Araucaria angustifolia, an endangered and native conifer from south Brazil. In this species SE includes the induction and proliferation of embryogenic cultures composed of pro-embryogenic masses (PEMs), which precede somatic embryos development. A. angustifolia SE model encompasses induction, proliferation, pre-maturation, and maturation steps. Double-staining with acetocarmine and Evan's blue is useful to evaluate the embryonic somatic structures. In this chapter we describe A. angustifolia SE protocols and analyzes morphological features in the different SE developmental stages.

The present study intended to investigate the effects of different glutathione (GSH) levels (0, 0.1, 0.5 and 1 mM) on the somatic embryogenesis (SE) induction of Acca sellowiana . Besides, we evaluated the effect of different carbon sources (sucrose and maltose) on the somatic embryos conversion. GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days of culture. The total number of somatic embryos obtained did not differ between treatments, but significant differences were observed for the embryonic stages after 80 days of culture. After 80 days of culture, 0.5 and 1 mM GSH-supplemented treatments showed the largest amount of torpedo-staged somatic embryos. In contrast, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages. These results indicate that GSH accelerates the SE induction process and increases the synchrony of the somatic embryo formation of A. sellowiana . The use of maltose for the somatic embryos conversion, as compared to sucrose, did not influence the conversion rate of normal chlorophyllous somatic embryos, but increased the formation of normal achlorophyllous somatic plantlets. This finding can be attributed to the rapid hydrolysis of sucrose, contributing to an enhanced chlorophyll synthesis.

Cattleya tigrina is endemic to the Atlantic forest biome and classified as vulnerable in the Red Book of Brazilian Flora. In vitro techniques comprise valuable tools for the conservation of endangered plant species. The aim of the present study was to evaluate the morphological features, global DNA methylation levels and free polyamines during protocorm-like bodies (PLBs) induction of C. tigrina . Along with that, an efficient protocol for in vitro propagation of this species is proposed. The first evidences of PLBs induction in C. tigrina occurred at seven days in culture, starting from the basal portion of the leaf abaxial surface. A hypomethylation marked the beginning of cell differentiation, followed by an increased global DNA methylation at 35 days in culture, coinciding with a subtle change in the structures morphogenetic development. During PLBs induction, putrescine exhibited higher levels as compared to spermidine and spermine, and apparently presents a major role during the PLBs induction in C. tigrina . Due to the apparent secondary PLBs formation, this protocol can represent a highly efficient method for in vitro propagation of this species.

Previous results have shown that an oxidizing product of firefly luciferin, dehydroluciferyl-adenylate, is the main intermediate in the process of synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase (EC 1.13.12.7). However, we have found that the pH effects on the luciferase oxidizing processes and on the synthesis of dinucleoside polyphosphate are opposite: acidic assay media enhance the synthesis of dinucleoside polyphosphate and inhibit the oxidizing processes. The reason for this apparent contradiction lies on the activation effect of low pH on the adenylate transfer reaction from dehydroluciferyl-adenylate to the acceptor nucleotide.