A semi-quantitative histochemical method for assessment of biochemical responses to osmotic stress in Coffea arabica leaf disks.

A simple method set for assessing biochemical changes associated with osmotic stress responses was developed using coffee (Coffea arabica L.) leaf disks. Stress was induced by polyethylene glycol (PEG) exposure. Quantitative evaluation of tissue physiological stress parameters was carried out using analytical methods to validate the conversion of classic qualitative histochemical tests for localizing lipid peroxidation, hydrogen peroxide, and total xanthine alkaloids into semi-quantitative assays. Relative electrolyte leakage (EL%) and chlorophyll content (SPAD index) were also recorded. EL% levels of treated disks were higher than those of control ones, whereas SPAD indexes were comparable. Histochemical localization indicated that levels of lipid peroxidation, H2O2, and total xanthines were also higher under osmotic stress than in control conditions. Semi-quantitative data obtained by image processing of histochemical staining consistently matched quantitative evaluations. Chromatographic analyses revealed that theophylline and caffeine concentrations increased in the presence of PEG, whereas theobromine remained constant in relation to the control. The methods herein described can be useful to rapidly acquire initial data regarding biochemical osmotic stress responses in coffee tissues based on simple staining and imaging steps. Moreover, it is likely that the same method may be applicable to other types of stresses and plant species upon minor adjustments.

Carvacrol-loaded nanoemulsions produced with a natural emulsifier for lettuce sanitization.

Carvacrol is an antimicrobial agent that shows potential for eliminating microorganisms in vegetables, increasing food safety. However, intense odor and low water solubility of carvacrol are limiting factors for its application for fresh vegetables sanitization, which can be overcome by nanotechnology. Two different nanoemulsions containing carvacrol (11 mg/mL) were developed by probe sonication: carvacrol-saponin nanoemulsion (CNS) and carvacrol-polysorbate 80 nanoemulsion (CNP). Formulations presented appropriate droplet sizes (from 74.7 nm to 168.2 nm) and high carvacrol encapsulation efficiency (EE) (from 89.5 % to 91.5 %). CNS showed adequate droplet size distribution (PDI < 0.22) and high zeta potential values (around -30 mV) compared to CNP, with saponin chosen for the following experiments. Carvacrol nanoemulsions presented Bacterial Inactivation Concentration (BIC) against the Salmonella cocktail from 5.51 to 0.69 mg/mL and for the E. coli cocktail from 1.84 to 0.69 mg/mL. Among all tested nanoemulsions, CNS1 presented the lowest BIC (0.69 mg/mL) against both bacterial cocktails. Damage to bacterial cells in lettuce treated with nanoemulsion was confirmed by scanning electron microscopy. For lettuce sanitization, CNS1 showed a similar effect to unencapsulated carvacrol, with a high bacterial reduction (>3 log CFU/g) after lettuce immersion for 15 min at 2 × BIC. Using the same immersion time, the CNS1 (2 × BIC) demonstrated equal or better efficacy in reducing both tested bacterial cocktails (>3 log CFU/g) when compared to acetic acid (6.25 mg/mL), citric acid (25 mg/mL), and sodium hypochlorite solution (150 ppm). Lettuce immersed in CNS1 at both concentrations (BIC and 2 × BIC) did not change the color and texture of leaves, while the unencapsulated carvacrol at 2 × BIC darkened them and reduced their firmness. Consequently, carvacrol-saponin nanoemulsion (CNS1) proved to be a potential sanitizer for lettuce.

Biochemical responses in leaf tissues of alkaloid producing Psychotria brachyceras under multiple stresses.

Under natural conditions plants are generally subjected to complex scenarios of combined or sequential environmental stresses. Among the various components of plant biochemistry modulated by abiotic variables, a pivotal role is played by antioxidant systems, including specialized metabolites and their interaction with central pathways. To help address this knowledge gap, a comparative analysis of metabolic changes in leaf tissues of the alkaloid accumulating plant Psychotria brachyceras Müll Arg. under individual, sequential, and combined stress conditions was carried out. Osmotic and heat stresses were evaluated. Protective systems (accumulation of the major antioxidant alkaloid brachycerine, proline, carotenoids, total soluble protein, and activity of the enzymes ascorbate peroxidase and superoxide dismutase) were measured in conjunction with stress indicators (total chlorophyll, ChA/ChB ratio, lipid peroxidation, H2O2 content and electrolyte leakage). Metabolic responses had a complex profile in sequential and combined stresses compared to single ones, being also modified over time. Different stress application schemes affected alkaloid accumulation in distinct ways, exhibiting similar profile to proline and carotenoids, constituting a complementary triad of antioxidants. These complementary non-enzymatic antioxidant systems appeared to be essential for mitigating stress damage and re-establishing cellular homeostasis. The data herein provides clues that may aid the development of a key component framework of stress responses and their appropriate balance to modulate tolerance and yield of target specialized metabolites.

Carbon Sequestration in Resin-Tapped Slash Pine (Pinus elliottii Engelm.) Subtropical Plantations.

Every year more than 150,000 tons of resin used in a myriad of industrial applications are produced by Brazilian plantations of Pinus elliottii Engelm. (slash pine), which are also used for timber. A pine tree can be tapped for resin over a period of several years. Resin is a complex mixture of terpenes, which are carbon-rich molecules, presumably influencing pine plantation carbon budgets. A total of 270 trees (overall mean DBH of 22.93 ± 0.11 cm) of 14-, 24-, and 26-year-old stands had their C content measured. Three different treatments (intact, wounded panels, and wounded + chemically stimulated panels, 30 trees each) were applied per site. Above- and belowground biomass, as well as resin yield, were quantified for two consecutive years. Data were statistically evaluated using normality distribution tests, analyses of variance, and mean comparison tests (p ≤ 0.05). The highest resin production per tree was recorded in the chemically stimulated 14-year-old stand. Tree dry wood biomass, a major stock of carbon retained in cell wall polysaccharides, ranged from 245.69 ± 11.73 to 349.99 ± 16.73 kg among the plantations. Variations in carbon concentration ranged from 43% to 50% with the lowest percentages in underground biomass. There was no significant difference in lignin concentrations. Soils were acidic (pH 4.3 ± 0.10-5.83 ± 0.06) with low C (from 0.05% to 1.4%). Significantly higher C stock values were recorded in pine biomass compared to those reported for temperate zones. Resin-tapping biomass yielded considerable annual increments in C stocks and should be included as a relevant component in C sequestration assessments of planted pine forests.

Methyl jasmonate induces selaginellin accumulation in Selaginella convoluta.

INTRODUCTION: Selaginellins are specialized metabolites and chemotaxonomic markers for Selaginella species. Despite the growing interest in these compounds as a result of their bioactivities, they are accumulated at low levels in the plant. Hence, their isolation and chemical characterization are often difficult, time consuming, and limiting for biological tests. Elicitation with the phytohormone methyl jasmonate (MeJA) could be a strategy to increase the content of selaginellins addressing their low availability problem, that also impairs pharmacological investigations. MATHERIALS AND METHODS: In this study, we examined MeJA elicitation in Selaginella convoluta plants, a medicinal plant found in northeastern Brazil, by treating them with two different concentrations (MeJA: 50 and 100 µM), followed by chemical profiling after 12, 24 and 48 h after application. Samples were harvested and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS AND DISCUSSCION: MeJA treatment significantly impacted the chemical phenotype. Regarding shoots differences in the time-dependent increased accumulation of all metabolites when plants were subjected to 100 µM MeJA were observed while in roots, most metabolites had their concentrations decreased in a time-dependent fashion at the same conditions. Results support organ, MeJA concentration and time post-treatment dependence of specialized metabolite accumulation, mainly the flavonoids and selaginellins. The amount of Selaginellin G in shoots of MeJA-treated specimens increased in 5.63-fold relative to control. The molecular networking approach allowed for the putative annotation of 64 metabolites, among them, the MeJA treatment followed by targeted metabolome analysis also allowed to annotate seven unprecedented selaginellins. Additionally, the in silico bioactive potential of the annotated selaginellins highlighted targets related to neurodegenerative disorders, antiproliferative, and antiparasitic issues. Taken together, data point out MeJA exposure as a strategy to induce potentially bioactive selaginellins accumulation in S. convoluta, this approach could enable a deep investigation about the metabolic function of these metabolites in the genus as well as regarding pharmacological exploration of the undervalued potential.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

Histochemical Localization of Phenolic Compounds and Reactive Oxygen Species in Eucalypt Microcuttings.

Among the main features of plant specialized metabolism are cell- and tissue- specific expression and responsiveness to oxidative stress conditions. Although quantitative techniques have significantly improved over time, allowing higher levels of resolution in plant metabolic studies, such analyses are often expensive and/or require relatively large amounts of starting material. The following protocols offer a relatively simple way to survey specialized and related metabolites (total phenolics, flavonoids, lignins), as well as reactive oxygen species (superoxide and hydrogen peroxide), using light and fluorescence microscopy. Also, a step-by-step guide on how to quantify stained areas is provided. Through the association of qualitative and quantitative data, general patterns of molecule distribution across plant tissues may be inferred, allowing for the solving of biological questions, and contributing to hypothesis refinement. Eucalyptus microcuttings were chosen as plant material to exemplify how these protocols can provide useful data for the understanding of complex developmental processes, such as adventitious root formation, which may be influenced by specialized metabolites and redox conditions.

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves.

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets for modulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga.

Molecular Networking Discloses the Chemical Diversity of Flavonoids and Selaginellins in Selaginella convoluta.

Selaginella convoluta is a desiccation tolerant plant native to the Brazilian semiarid region (Caatinga), endowed with an effective drought resistance mechanism. As part of our research efforts to understand the chemical diversity of S. convoluta, dehydrated (harvested in their natural habitat in the dry season) and hydrated (plant acclimated in a laboratory after rehydration) specimens were analyzed by HR-LC-ESI-MS/MS followed by a structural annotation on the Global Natural Products Social Molecular Networking Web platform. The molecular networking approach allowed for putative annotation of 39 metabolites, mainly selaginellins and flavonoids. Based on MS/MS data, three unprecedented selaginellins were annotated: 29-hydroxy selaginellin O, 29-hydroxy selaginellin A, and 4-{[2-(4-hydrophenyl)-6-[2-(4-hydroxyphenyl)ethynyl]phenyl](4-oxocyclohexa-2,5-dien-1-ylidene)methyl}benzaldehyde. Th results pointed out that valuable scientific knowledge can be obtained from studies conducted with plants in their natural habitat by allowing a more realistic profile of chemical diversity. The present study adds new information on specialized metabolites of S. convoluta, mainly flavonoids and selaginellins, and highlights the species as an untapped source of chemobiodiversity from Caatinga.

Erythroxylum pungens Tropane Alkaloids: GC-MS Analysis and the Bioactive Potential of 3-(2-methylbutyryloxy)tropan-6,7-diol in Zebrafish (Danio rerio).

Tropane alkaloids are specialized plant metabolites mostly found in the Erythroxylaceae and Solanaceae families. Although tropane alkaloids have a high degree of structural similarity because of the tropane ring, their pharmacological actions are quite distinct. Brazil is one of the main hotspots of Erythroxylum spp. diversity with 123 species (almost 66% of the species catalogued in tropical America). Erythroxylum pungens occurs in the Caatinga, a promising biome that provides bioactive compounds, including tropane alkaloids. As part of our efforts to investigate this species, 15 alkaloids in specimens harvested under different environmental conditions are presented herein. The occurrence of 3-(2-methylbutyryloxy)tropan-6,7-diol in the stem bark of plants growing in their natural habitat, greenhouse controlled conditions, and after a period of water restriction, suggests that it is a potential chemical marker for the species. This alkaloid was evaluated for several parameters in zebrafish (Danio rerio) as a model organism. Regarding toxicity, teratogenic effects were observed at 19.5 µM and the lethal dose for embryos was 18.4 µM. No mortality was observed in adults, but a behavioral screen showed psychostimulatory action at 116.7 µM. Overall, the alkaloid was able to cause zebrafish behavioral changes, prompting further investigation of its potential as a new molecule in the treatment of depression-like symptoms. In silico, targets involved in antidepressant pathways were identified by docking.

Microcutting Redox Profile and Anatomy in Eucalyptus spp. With Distinct Adventitious Rooting Competence.

Adventitious root (AR) development takes place in an intricate cellular environment. Reactive oxygen species (ROS) and antioxidant defenses, triggered by wounding in cuttings, can modulate this process. A comparative assessment of biochemical and anatomical parameters at critical rooting stages in hard- (Eucalyptus globulus Labill.) and easy- (Eucalyptus grandis W.Hill ex Maiden) to-root species was carried out. Microcuttings from seedlings were inoculated in auxin-free AR induction medium and, after 96 h, transferred to AR formation medium for a period of 24 h. Samples were collected upon excision (Texc) and at the 5th day post excision (Tform). Delayed xylem development, with less lignification, was recorded in E. globulus, when compared to E. grandis, suggesting lower activity of the cambium layer, an important site for AR development. Superoxide was more densely present around the vascular cylinder at both sampled times, and in greater quantity in E. globulus than E. grandis, declining with time in the former. Hydrogen peroxide was localized primarily along cell walls, more intensely in the primary xylem and phloem, and increased significantly at Tform in E. globulus. Ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities were generally higher in E. grandis and varied as a function of time in E. globulus. Soluble guaiacol peroxidase (GPRX) activity increased from Texc to Tform in both species, whereas cell wall-bound GPRX activity increased with time in E. grandis, surpassing E. globulus. Flavonoid content increased with time in E. grandis and was higher than E. globulus at Tform. Principal component analysis showed that species- and time-derived differences contributed to almost 80% of the variance. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.

The role of auxin transporters and receptors in adventitious rooting of Arabidopsis thaliana pre-etiolated flooded seedlings.

Adventitious roots (ARs) form from above-ground organs, and auxins are major regulators of AR development. TIR1/AFB F-box proteins act as well-established auxin receptors. Auxin transport involves the PINFORMED (PIN) auxin efflux carriers and AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX1) influx carriers. To further elucidate the basis of AR development, we investigated the participation of these proteins and phosphorylation of PINs during adventitious rooting in hypocotyls of pre-etiolated flooded Arabidopsis thaliana seedlings. Mutant and GUS localization studies indicated that AFB2 is important in AR development. AUX1 loss-of-function reduced AR numbers, which could not be reversed by exogenous auxin. Single mutations in LAX1, LAX2 and LAX3 had no negative impact on AR development and the first and last mutations even promoted it. Double and triple mutants of AUX1, LAX1, LAX2 and LAX3 significantly reduced rooting, which was reversed by exogenous auxin. AUX1 was essential in AR establishment, with LAX3 apparently acting in conjunction. Proper phosphorylation of PINs by PID, WAG1 and WAG2 and auxin transport direction were equally essential for AR establishment. PIN1, AUX1 and AFB2 (overexpression) and LAX1, LAX3, PIN4 and PIN7 (downregulation) emerged as potential targets for genetic manipulation aiming at improving AR development.

Mimosine accumulation in Leucaena leucocephala in response to stress signaling molecules and acute UV exposure.

Mimosine is a non-protein amino acid of Fabaceae, such as Leucaena spp. and Mimosa spp. Several relevant biological activities have been described for this molecule, including cell cycle blocker, anticancer, antifungal, antimicrobial, herbivore deterrent and allelopathic activities, raising increased economic interest in its production. In addition, information on mimosine dynamics in planta remains limited. In order to address this topic and propose strategies to increase mimosine production aiming at economic uses, the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation in growth room-cultivated seedlings of Leucaena leucocephala spp. glabrata. Mimosine concentration was not significantly affected by 10 ppm salicylic acid (SA) treatment, but increased in roots and shoots of seedlings treated with 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound), and in shoots treated with UV-C radiation. Quantification of mimosine amidohydrolase (mimosinase) gene expression showed that ethephon yielded variable effect over time, whereas JA and UV-C did not show significant impact. Considering the strong induction of mimosine accumulation by acute UV-C exposure, additional in situ ROS localization, as well as in vitro antioxidant assays were performed, suggesting that, akin to several secondary metabolites, mimosine may be involved in general oxidative stress modulation, acting as a hydrogen peroxide and superoxide anion quencher.

Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology.

Species of Eucalyptus are some of the most planted trees in the world, providing fiber, cellulose, energy, and wood for construction and furniture in renewable fashion, with the added advantage of fixing large amounts of atmospheric carbon. The efficiency of eucalypts in forestry relies mostly on the clonal propagation of selected genotypes both as pure species and interspecific hybrids. The formation of new roots from cambium tissues at the base of cuttings, referred to as adventitious rooting (AR), is essential for accomplishing clonal propagation successfully. AR is a highly complex, multi-level regulated developmental process, affected by a number of endogenous and environmental factors. In several cases, highly desirable genotypes from an industrial point of view carry along the undesirable trait of difficulty-to-root (recalcitrance). Understanding the bases of this phenotype is needed to identify ways to overcome recalcitrance and allow efficient clonal propagation. Herein, an overview of the state-of-the-art on the basis of AR recalcitrance in eucalypts addressed at various levels of regulation (transcript, protein, metabolite and phenotype), and OMICs techniques is presented. In addition, a focus is also provided on the gaps that need to be filled in order to advance in this strategic biological problem for global forestry industry relying on eucalypts.

Quillaja brasiliensis saponin-based nanoparticulate adjuvants are capable of triggering early immune responses.

Commercially available saponins are extracted from Quillaja saponaria barks, being Quil A® the most widely used. Nanoparticulate immunostimulating complexes (ISCOMs or ISCOMATRIX) formulated with these, are able to stimulate strong humoral and cellular immune responses. Recently, we formulated novel ISCOMs replacing QuilA® by QB-90 (IQB-90), a Quillaja brasiliensis leaf-extracted saponin fraction, and reported that IQB-90 improved antigen uptake, and induced systemic and mucosal antibody production, and T-cell responses. However, its mechanism of action remains unclear. In this study we provide a deeper insight into the immune stimulatory properties of QB-90 and ISCOMATRIX-like based on this fraction (IMXQB-90). We show herein that, when used as a viral vaccine adjuvant, QB-90 promotes an "immunocompetent environment". In addition, QB-90 and IMXQB-90 induce immune-cells recruitment at draining-lymph nodes and spleen. Subsequently, we prove that QB-90 or IMXQB-90 stimulated dendritic cells secret IL-1ß by mechanisms involving Caspase-1/11 and MyD88 pathways, implying canonical inflammasome activation. Finally, both formulations induce a change in the expression of cytokines and chemokines coding genes, many of which are up-regulated. Findings reported here provide important insights into the molecular and cellular mechanisms underlying the adjuvant activity of Q. brasiliensis leaf-saponins and its respective nanoparticles.

Comparative adventitious root development in pre-etiolated and flooded Arabidopsis hypocotyls exposed to different auxins.

Adventitious roots (ARs) emerge from stems, leaves or hypocotyls, being strategic for clonal propagation. ARs may develop spontaneously, upon environmental stress or hormonal treatment. Auxins strongly influence AR development (ARD), depending on concentration and kind. However, the role of different types of auxin is rarely compared at the molecular level. Rooting triggered by light exposure and flooding was examined in intact etiolated Arabidopsis thaliana hypocotyls treated with distinct auxin types. Morphological aspects, rooting-related gene expression profiles, and IAA immunolocalization were recorded. NAA and 2,4-D effects were highly dose-dependent; at higher concentrations NAA inhibited root growth and 2,4-D promoted callus formation. NAA yielded the highest number of roots, but inhibited elongation. IAA increased the number of roots with less interference in elongation, yielding the best overall rooting response. IAA was localized close to the tissues of root origin. Auxin stimulated ARD was marked by increased expression of PIN1 and GH3.3. NAA treatment induced expression of CYCB1, GH3.6 and ARF8. These NAA-specific responses may be associated with the development of numerous shorter roots. In contrast, expression of the auxin action inhibitor IAA28 was induced by IAA. Increased PIN1 expression indicated the relevance of auxin efflux transport for focusing in target cells, whereas GH3.3 suggested tight control of auxin homeostasis. IAA28 increased expression during IAA-induced ARD differs from what was previously reported for lateral root development, pointing to yet another possible difference in the molecular programs of these two developmental processes.

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

The role of Zn2+, dimerization and N-glycosylation in the interaction of Auxin-Binding Protein 1 (ABP1) with different auxins.

Auxin is critical for plant growth and development. The main natural auxin is indole-3-acetic acid (IAA), whereas 1-naphthalene acetic acid (NAA) is a synthetic form. Auxin-Binding Protein 1 (ABP1) specifically binds auxins, presumably playing roles as receptor in nontranscriptional cell responses. ABP1 structure was previously established from maize at 1.9 Å resolution. To gain further insight on ABP1 structural biology, this study was carried out employing molecular dynamics simulations of the complete models of the oligomeric glycosylated proteins from maize and Arabidopsis thaliana with or without auxins. In maize, both Zn2+ coordination and glycosylation promoted conformational stability and most of such stabilization effect was located on the N-terminal region. The α-helix of C-terminal regions in ABP1 of both species unfolded during simulations, assuming a more extended structure in maize. In Arabidopsis, the helix appeared more stable, being preserved in most of the monomeric simulations and unfolding when the protein was in the dimeric form. In Arabidopsis ABP1 bound to IAA or NAA, glycosylation structures arranged around the protein, covering the putative site of entrance or egress of auxin. NAA bound protein folding was more similar to the crystal structure showing higher stability compared to that of IAA bound. The molecular structural differences of ABP1 found between the species and auxin types indicate that this auxin-binding protein shows functional specificities in dicots and monocots, as well as in auxin type binding.

Quillaja brasiliensis saponins induce robust humoral and cellular responses in a bovine viral diarrhea virus vaccine in mice.

A saponin fraction extracted from Quillaja brasiliensis leaves (QB-90) and a semi-purified aqueous extract (AE) were evaluated as adjuvants in a bovine viral diarrhea virus (BVDV) vaccine in mice. Animals were immunized on days 0 and 14 with antigen plus either QB-90 or AE or an oil-adjuvanted vaccine. Two-weeks after boosting, antibodies were measured by ELISA; cellular immunity was evaluated by DTH, lymphoproliferation, cytokine release and single cell IFN-γ production. Serum anti-BVDV IgG, IgG1 and IgG2b were significantly increased in QB-90- and AE-adjuvanted vaccines. A robust DTH response, increased splenocyte proliferation, Th1-type cytokines and enhanced production of IFN-γ by CD4(+) and CD8(+) T lymphocytes were detected in mice that received QB-90-adjuvanted vaccine. The AE-adjuvanted preparation stimulated humoral responses but not cellular immune responses. These findings reveal that QB-90 is capable of stimulating both cellular and humoral immune responses when used as adjuvant.

Comparative transcriptional analysis provides new insights into the molecular basis of adventitious rooting recalcitrance in Eucalyptus.

Adventitious rooting (AR) is essential in clonal propagation. Eucalyptus globulus is relevant for the cellulose industry due to its low lignin content. However, several useful clones are recalcitrant to AR, often requiring exogenous auxin, adding cost to clonal garden operations. In contrast, E. grandis is an easy-to-root species widely used in clonal forestry. Aiming at contributing to the elucidation of recalcitrance causes in E. globulus, we conducted a comparative analysis with these two species differing in rooting competence, combining gene expression and anatomical techniques. Recalcitrance in E. globulus is reversed by exposure to exogenous indole-3-acetic acid (IAA), which promotes important gene expression modifications in both species. The endogenous content of IAA was significantly higher in E. grandis than in E. globulus. The cambium zone was identified as an active area during AR, concentrating the first cell divisions. Immunolocalization assay showed auxin accumulation in cambium cells, further indicating the importance of this region for rooting. We then performed a cambium zone-specific gene expression analysis during AR using laser microdissection. The results indicated that the auxin-related genes TOPLESS and IAA12/BODENLOS and the cytokinin-related gene ARR1may act as negative regulators of AR, possibly contributing to the hard-to-root phenotype of E. globulus.