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Plant micropropagation comprises biotechnological tools used for the conservation and mass propagation of orchid species. However, there are few reports of its use for orchids of the subtribe Pleurothallidinae. The present study evaluated the effects of 6-benzylaminopurine (BAP) on shoots formation and development of Dryadella zebrina (Porsch) Luer. For this, seeds were in vitro germinated, and the plantlets were submitted to different concentrations of BAP (0, 3, 6, 9, 12, and 15 µM). The plantlets derived from the treatments 0, 6, and 15 µM of BAP were collected after 60 days in culture and subjected to light microscopy analysis. Our results indicated that BAP increased the formation of the new shoots of D. zebrina, especially in treatment with 6 µM, and its use is indicated for the in vitro multiplication phase. The anatomical analyses of the roots showed a deleterious effect of 15 µM BAP on the meristematic region, with the presence of more vacuolated cells in this zone. Our results represent the first successful report of in vitro propagation for the genus Dryadella and may serve as a basis for further studies of in vitro propagation of phylogenetically related species.

Somatic embryogenesis (SE) involves modifications of cellular, biochemical, genetic, and epigenetic patterns. Our work investigated proteins as markers of embryogenic response and characterized the redox state of embryogenic cultures (EC) of Guadua chacoensis. We identified a total of 855 proteins; 129 were up- and 136 down-accumulated in EC as compared with non-embryogenic culture (NEC). Additionally, 37 and 22 proteins were identified as unique in EC and NEC, respectively. Heat-shock proteins as unique proteins and increased activity in Superoxide Dismutase and Guaiacol Peroxidase in EC suggest that the embryogenic response requires activation of the stress response mechanism. Ribosomal, translational, and glycolytic proteins in EC seem to be associated with protein synthesis and energy sources for embryo development, respectively. Accumulation of cell wall-related proteins, such as Arabinogalactan and Polygalacturonase inhibitors, and signaling transduction proteins, including Chitinase, Phospholipase, and Guanine nucleotide-binding proteins in EC seems to be associated with embryogenic response. Enhancement of H

Here we sequenced and characterized the complete plastome of Bactris riparia, a species closely related to B. gasipaes and widely distributed in Western Amazonia. We performed a comparative genomic analysis with B. riparia and the other four Bactridinae species retrieved from GenBank. The plastome of B. riparia was 156,715 bp with a quadripartite structure. Gene content included 86 protein-coding genes (CDS), 38 tRNAs, and 8 rRNAs. Bactris riparia has 69 more base pairs than B. gasipaes, with identical numbers in IR, and more in LSC and SSC. The comparative analysis indicated that structure, collinearity, and IR/SSC borders of plastomes within subtribe Bactridinae are, in general, conserved. We predicted 69 SSRs in B. riparia plastome. Among them, ~80% consisted of A/T homopolymers. Among the 52 variable CDS, rbcL showed the highest non-synonymous rate, while the rps15 gene had the highest synonymous rate. Three genes (ccsA, cemA, and rpoC1) presented evidence of positive selection and 22 genes showed evidence of purifying selection. The phylogenetic tree based on plastome sequences set Bactris as more closely related to Astrocaryum than to Acrocomia. These new plastome data of B. riparia will contribute to studies about the diversity, evolutionary history, and conservation of palms.

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.

Bamboos are one of the largest Poaceae groups and comprise about 1600 species distributed worldwide. Many of these species are sources of food, handicrafts, construction, charcoal, paper, and fiber. However, the obtainment of financially viable and high-quality seedlings of bamboo is not an easy task. Micropropagation has been successfully used to obtain large-scale bamboo seedlings and is one of the most promising techniques to overcome the bottlenecks in the propagation of these species. Two morphogenetic routes are being used in bamboo micropropagation: organogenesis and somatic embryogenesis (SE). In SE, one or more somatic cells differentiate into somatic embryos, resembling the structure of the zygotic embryo, with bipolar axes that are not vascular connected with the source tissue. Historically, SE has been neglected in comparison to organogenesis, possibly due to its greater complexity and difficulty in control and modulation. So far, ~30 bamboo species have established SE protocols, most of which from genera Bambusa and Dendrocalamus. This chapter discusses the principal advances in bamboo SE, with emphasis on complementary approaches as morpho-anatomy, biochemistry, and molecular biology. Also, we outline the main constraints and limitations of this promising technique and new paths to develop more efficient protocols.

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.

Bambusa oldhamii Munro is a fast-growing species of woody bamboo with strong commercial appeal. In Brazil, the use of this species is limited, mainly due to the low availability of seedlings for commercial plantations. Micropropagation is a technique used for the large scale production of seedlings, but protocols for the establishment of aseptic cultures are hampered by the presence of endophytic contamination. This study aimed to develop an in vitro establishment protocol for B. oldhamii, as well as to make the molecular identification of fungi associated with the explants used. Nodal segments of adult plants grown in the field were used as explants. This material was submitted to two experiments carried out to evaluate the effect of 6-benzylaminopurine (BAP) on multiplication and of Plant Preservative Mixture (PPMTM) as a disinfectant. In the first one, 10 µM, 15 µM or 20 µM of BAP were combined with 1 mL L-1, 2 mL L-1 or 3 mL L-1 of PPMTM; while the second one used 0 µM, 2.5 µM, 5 µM or 7.5 µM of BAP with 4 mL L-1 of PPMTM, both added to MS culture medium. After 21 days of culture, the use of 4 mL L-1 of PPMTM inhibited the bacterial growth and reduced fungal contamination. The addition of BAP to the culture medium above 10 µM inhibited the formation and growth of new shoots, while additions of less than 7,5 µM had no effect. The molecular identification of the endophytic fungi isolated during the in vitro culture indicated the presence of numerous fungal species, increasing the current knowledge about the diversity of fungi associated with bamboo.

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).

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.

P. lambertii is a Brazilian conifer indigenous to one of the 25 biodiversity hotspots of the world, the subtropical moist forest ecoregion of the Atlantic Forest Biome. Its conservation status is classified as near threatened, being desirable the development and use of biotechnological tools, such as somatic embryogenesis (SE). SE is a complex, multifactorial model system of molecular, biochemical, physiological and developmental steps of plant embryogenesis, and 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. Protocols for plant regeneration based on SE have been established for many conifers species, but species from the closely related families Araucariaceae and Podocarpaceae are in general considered recalcitrant to SE. Mostly of the SE protocols for conifers have been developed for members of family Pinaceae. In this context, here we describe the first SE protocol for a species from family Podocarpaceae, the P. lambertii. This protocol is based on induction with immature zygotic embryos, followed by establishment and multiplication of proembryogenic masses on plant growth regulator-free culture medium, and maturation phases I and II. Procedures including explant preparation, PEM proliferation, and somatic embryo development are described in detail.

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.

Micropropagation of banana by means of different morphogenetic routes is an important source of genetically uniform, pest- and disease-free planting materials of Musa spp. Morphogenesis of in vitro grown plants are affected by light quality, light quantity and photoperiod. Light-emitting diode (LED) lighting systems have several unique advantages, including their peak spectral output, which closely coincides with the red absorption peak of chlorophyll and the reported wavelengths for maximum photosynthetic efficiency. The present study was aimed to investigate the effect of two LED lighting treatments (white LED and deep red/white LED) compared to conventional fluorescent lamps on the stomata formation and chlorophyll (Chl) levels in micropropagated banana plantlets through organogenesis. Both LED lighting evaluated increased the levels of total Chl, Chl-a and Chl-b in banana in vitro plantlets, indicating no statistical difference between them, but being higher then plantlets subjected to fluorescent lamps. The number of stomata was also increased equally by both LED lighting treatments, indicating increased formation of stomata in both abaxial and adaxial leaf surfaces when compared to fluorescent lamps. Although all treatments presented 100 % survival in the acclimatization phase, LED lighting show promising for micropropagation of banana due to its known superior light quality, low energy consumed, little heat generated and long operating lifetime.

Pineapple presents a significant portion of the global fruit market. Recently, a micropropagation protocol was established based on nodule cluster cultures (NC), which showed high regenerative efficiency. Aiming to expand the understanding of gene regulation in this protocol, global DNA methylation levels (GDML) and fresh mass increase ratio (FMIR) during NC multiplication and differentiation to microshoots were evaluated after 0, 9, 18, 27, and 36 days in culture. In addition, genetic fidelity of randomly chosen regenerated plants was analyzed by means of amplified fragment length polymorphism (AFLP), flow cytometric analysis of ploidy level, as well as observation of phenotypic features. An association between GDML and the density of new shoot apical meristems formed in cultures was observed, which was, in turn, related to FMIR and the age of each subculture. The analysis by means of AFLP and DNA ploidy level, as well as phenotypic observations indicated the homogeneity of regenerated plantlets.

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.

Acca sellowiana is a native species to the southern region of Brazil, with secondary dispersion in Uruguay northeast. The potentially economic interest of this species motivated many fundamental and applied researches, such as micropropagation protocols development, including somatic embryogenesis (SE). Previous studies have reported the high frequency of off-type somatic embryos in A. sellowiana SE and low conversion efficiency, possibly due to this high off-type somatic embryos formation. In this sense, this study was aimed to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) in the induction and development of somatic embryos in direct and indirect SE through cytogenetic and DNA methylation analysis. The results of the present work reinforces the previous findings that 2,4-D plays a key role in the SE induction in A. sellowiana. However, the continuous presence of this auxin in the culture medium enhances the rate of off-type somatic embryos. This auxin also apparently affects the cells and chromosomes size in normal and off-type somatic embryos and DNA methylation patterns of the analyzed samples. These findings may indicate that the off-type somatic embryos formation may be related to genetic and/or epigenetic changes caused by long-term cultivation in A. sellowiana. The intermediate phase of callus also indicated increased formation of off-types, corroborating other studies that showed that indirect SE could cause increased formation of anomalies in regenerated plantlets.

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

Morphological disorders in a relevant portion of emerged somatic embryos have been a limiting factor in the true-to-type plantlet formation in Acca sellowiana. In this sense, the present study undertook a comparison between normal phenotype and off-type somatic plantlets protein profiles by means of the 2-D DIGE proteomics approach. Off-type and normal phenotype somatic plantlets obtained at 10 and 20 days conversion were evaluated. Results indicated 12 exclusive spots between normal and off-type plantlets at 10 days conversion, and 17 exclusive spots at 20 days conversion. Also at 20 days conversion, 4 spots were differentially expressed, up- or down-regulated. Two proteins related to carbohydrate metabolism were only expressed in off-types at 10 days conversion, suggesting a more active respiratory pathway. A vicilin-like storage protein was only found in off-types at 20 days conversion, indicating that plantlets may present an abnormality in the mobilization of storage compounds, causing reduced vigor in the development of derived plantlets. The presence of heat shock proteins were only observed during formation of normal phenotype somatic plantlets, indicating that these proteins may be involved in normal morphogenesis of plantlets formed. These new findings shed light on possible genetic or epigenetic mechanisms governing A. sellowiana morphogenesis.

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.

Biotechnology tools based on tissue culture techniques allow mass clonal propagation of plants with desired genetic and sanitary features. In the present work a pineapple micropropagation protocol was established based on nodule cluster cultures (NC) associated with different culture systems. Leaf segments from in vitro -grown plantlets cultivated in culture medium supplemented with 2 μM NAA and 8 μM 2-iP or BAP resulted in the highest rates of NC induction. NC fresh weight increased ratio was more efficient in a culture medium supplemented with 2 μM NAA and 2 μM BAP in twin-flasks temporary immersion system (TIS-TF). This culture medium was also efficient for the microshoots development in RITA ® temporary immersion system (TIS-RITA ® ). Microshoots elongation in permanent immersion system (PIS) was more efficient in a culture medium supplemented with 10 μM GA 3 . Shoots longer than 2 cm showed 92% survival rate when acclimatized. Free polyamine endogenous levels was measured and showed higher contents for the TIS-TF, followed by TIS-RITA ® , and PIS, respectively. Putrescine levels also followed this pattern, being absent in PIS, which, on the other hand, showed higher levels of spermine and spermidine. The results indicate the establishment of efficient protocol for pineapple micropropagation, with a high multiplication and regenerative rates and great potential for agriculture application. The biochemical analysis suggests that besides culture medium composition, flask design and culture system also drastically affects the plant's metabolism toward multiplication and plant regeneration.

Background TIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn2 + finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix. Methods Here we characterized the Zn2 + induced TIM15 folding integrating biophysical and computational approaches. Results TIM15 folding occurs from an essentially unstructured conformation to a Zn2 +-coordinated protein in a fast and markedly temperature-dependent process. Moreover, we demonstrate unambiguously that Zn2 + induced TIM15 folding is essential for its role as mtHsp70 chaperone since in the unstructured apo state TIM15 does not bind to mtHsp70 and is unable to prevent its aggregation. Molecular dynamics simulations help to understand the crucial role of Zn2 + in promoting a stable and functional 3D architecture in TIM15. It is shown that the metal ion, through its coordinating cysteine residues, can mediate relevant long-range effects with the interaction interface for mtHsp70 coupling thus folding and function. Conclusions Zn2 + induced TIM15 folding is essential for its function and likely occurs in mitochondrial matrix where high concentrations of Zn2 + were reported. General significance The combination of experimental and computational approaches presented here provide an integrated structural, kinetic and thermodynamic view of the folding of a mitochondrial zinc finger protein, which might be relevant to understand the organelle import of proteins sharing this fold.

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

Retrophyllum piresii (Podocarpaceae) is an endemic conifer species from the Brazilian Amazonian Region, and very few data related to ecological and genetic characteristics of this species are available. Plastome sequencing is an efficient tool to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as to probe the structural and functional evolution of plants. Usually, the plastome of photosynthetic land plants is quadripartite, with two copies of the inverted repeats (IRs) separating the small and large single-copy regions. However, in gymnosperms, IR can vary from large in size to completely absent, being constituted principally by transfer RNA (tRNA) genes, or a part of sequence of other genes. Here, we sequenced and characterized the complete plastome of R. piresii. This plastome was determined to be 133,291 bp (~480-fold coverage), presenting a total of 120 identified genes, of which 118 were single copy and two genes, trnN-GUU and trnD-GUC, were found to be duplicated and occurring as inverted and directed repeat (DR) sequences, respectively. These repeated regions presented recombinationally active sites, resulting in an IR-mediated inversion and a DR-mediated deletion. However, the isoform resulted from DR-mediated deletion may result in unviable plastome, with deletion of photosynthetic and expression machinery-related genes.

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.

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia.

Background Mono and dinucleoside polyphosphates (pnNs and NpnNs) exist in living organisms and induce diverse biological effects through interaction with intracellular and cytoplasmic membrane proteins. The source of these compounds is associated with secondary activities of a diverse group of enzymes. Scope of review Here we discuss the mechanisms that can promote their synthesis at a molecular level. Although all the enzymes described in this review are able to catalyse the in vitro synthesis of NpnNs (and/or pnN), it is not clear which ones are responsible for their in vivo accumulation. Major conclusions Despite the large amount of knowledge already available, important questions remain to be answered and a more complete understanding of pnNs and NpnNs synthesis mechanisms is required. With the possible exception of (GTP:GTP guanylyltransferase of Artemia), all enzymes able to catalyse the synthesis of pnNs and NpnNs are unspecific and the factors that can promote their synthesis relative to the canonical enzyme activities are unclear. General significance The fact that pnNs and NpnNs syntheses are promiscuous activities of housekeeping enzymes does not reduce its physiological or pathological importance. Here we resume the current knowledge regarding their enzymatic synthesis and point the open questions on the field.

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.

Vaccine and drug development for fasciolasis rely on a thorough understanding of the mechanisms involved in parasite-host interactions. FH8 is an 8 kDa protein secreted by the parasite Fasciola hepatica in the early stages of infection. Sequence analysis revealed that FH8 has two EF-hand Ca(2+)-binding motifs, and our experimental data show that the protein binds Ca(2+) and that this induces conformational alterations, thus causing it to behave like a sensor protein. Moreover, FH8 displays low affinity for Ca(2+) (K(obs) = 10(4) m(-1)) and is highly stable in its apo and Ca(2+)-loaded states. Homology models were built for FH8 in both states. It has only one globular domain, with two binding sites and appropriate groups in the positions for coordination of the metal ions. However, an unusually high content of positively charged amino acids in one of the binding sites, when compared with the prototypical sensor proteins, potentially affects the protein's affinity for Ca(2+). The only Cys present in FH8, conserved in the homologous proteins of other helminth parasites, is located on the surface, allowing the formation of dimers, detected on SDS gels. These findings reflect specificities of FH8, which are most probably related to its roles both in the parasite and in the host.