Phytotoxicity of Quillaja lancifolia Leaf Saponins and Their Bioherbicide Potential.

Weeds are major threats to the integrity of agricultural and natural environments due to their invasive and competing potential. Bioherbicides are substances based on natural compounds that are biodegradable and often have low residual effects. Plant species able to produce and release phytotoxic compounds may represent effective bioherbicide sources. Leaves of Quillaja lancifolia D.Don (formerly Q. brasiliensis (A.St.-Hil. & Tul.) Mart.) produce water-soluble specialized metabolites of the saponin class that could be evaluated for phytotoxic activity and potential as natural herbicides. This study was conducted to examine the impacts of Q. lancifolia total saponins aqueous extract (AE) at 4 and 10% (w/v) and of two combined reverse-phase chromatography purified saponin fractions (QB) at 1 and 2% (w/v) on morpho-physiological parameters of Lactuca sativa (lettuce) and Echinochloa crus-galli (barnyardgrass) in pre- and post-emergence bioassays. QB was only tested in pre-emergence assays. In pre-emergence bioassays, the germination rate and germination kinetics were determined. Post-emergence evaluations included effects on seedling morphology, root and shoot length, dry mass, and chlorophyll content. Osmotic potential and pH analyses ruled out roles for these factors in the observed responses. AE had a high inhibitory impact on the germination of both lettuce and barnyardgrass. QB at 1% and 2% (w/v) significantly decreased the growth of lettuce seedlings germinated in its presence by more than 10-fold. Phytotoxic effects on the post-emergence growth of lettuce, especially at the highest concentration tested of AE (10% w/v), was also observed. The presence of quillaic acid-based triterpene saponins in AE and QB was confirmed using different analytical methods. Therefore, both saponin-enriched fraction and aqueous extracts of Q. lancifolia inhibited tested plant growth and development. The water solubility of saponins and the availability of a sustainable source of these molecules from the leaves of cultivated young Q. lancifolia plants make them attractive candidates for use as bioherbicides.

Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.

Clonal Eucalyptus propagation is essential for various industry sectors. It requires cuttings to successfully develop adventitious roots (ARs). Environmental conditions are influential on AR development and may be altered to modulate the productivity of hard-to-root clones. The current knowledge gap in research on the physiological patterns underlying commercial-scale propagation results hinders the design of novel strategies. This study aimed to identify patterns of variation in AR-relevant parameters in contrasting seasons and species with distinct rooting performances. E. dunnii and E. ×urograndis (hard- (hardR) and easy-to-root (easyR), respectively) mini-stumps were subjected to light modulation treatments and to mini-tunnel use (MT) for a year. The treatment impact on the branching and rooting rates was recorded. The carbohydrate content, AR-related gene expression, and mineral nutrition profiles of cuttings from the control (Ctrl) and treated mini-stumps were analyzed. Light treatments were often detrimental to overall productivity, while MTs had a positive effect during summer, when it altered the cutting leaf nutrient profiles. Species and seasonality played large roles in all the assessed parameters. E. ×urograndis was particularly susceptible to seasonality, and its overall superior performance correlated with changes in its gene expression profile from excision to AR formation. These patterns indicate fundamental differences between easyR and hardR clones that contribute to the design of data-driven management strategies aiming to enhance propagation protocols.

Methods of Mimosine Extraction from Leucaena leucocephala (Lam.) de Wit Leaves.

Mimosine is a nonprotein amino acid biosynthesized from OAS (O-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3,4-dihydroxypyridine). This amino acid constitutively occurs in all parts of Leucaena leucocephala (Lam.) de Wit plants and is found at higher concentrations in seeds and leaves. This metabolite has several useful activities, such as antioxidant, allelochemical, insecticidal, antimicrobial, metal chelating, and antitumor. Mimosine is well studied in biomedical research due its ability to inhibit cells in the late G1 phase and to induce cell apoptosis. Two simple methods of mimosine extraction from leucaena leaves, pulverized and whole maceration, are described herein in detail.

Foamy matters: an update on Quillaja saponins and their use as immunoadjuvants.

Immunoadjuvant Quillaja spp. tree saponins stimulate both cellular and humoral responses, significantly widening vaccine target pathogen spectra. Host toxicity of specific saponins, fractions and extracts may be rather low and further reduced using lipid-based delivery systems. Saponins contain a hydrophobic central aglycone decorated with several sugar residues, posing a challenge for viable chemical synthesis. These, however, may provide simpler analogs. Saponin chemistry affords characteristic interactions with cell membranes, which are essential for its mechanism of action. Natural sources include Quillaja saponaria barks and, more recently, Quillaja brasiliensis leaves. Sustainable large-scale supply can use young plants grown in clonal gardens and elicitation treatments. Quillaja genomic studies will most likely buttress future synthetic biology-based saponin production efforts.

Corrigendum: Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis.

[This corrects the article on p. 849 in vol. 7, PMID: 27379135.].

Events Associated with Early Age-Related Decline in Adventitious Rooting Competence of Eucalyptus globulus Labill.

The development of adventitious roots is affected by several factors, including the age of the cutting donor plant, which negatively affects rooting capacity. Eucalyptus globulus quickly loses rooting capacity of cuttings as the donor plant ages, although the molecular and biochemical mechanisms behind this process are still unclear. To better understand the bases of rooting competence loss in E. globulus, the time required for a significant decline in rhizogenic ability without exogenous auxin was determined in microcuttings derived from donor plants of different ages after sowing. Tip cuttings of donor plants were severed before and after loss of rooting competence of microcuttings to test the hypothesis that auxin and carbohydrate homeostasis regulate rooting competence decline. There were no significant changes in concentration of carbohydrates, flavonoids, or proteins before and after the loss of rooting capacity. Peroxidase (EC 1.11.1.7) total activity increased with loss of rooting competence. Auxin concentration showed the opposite pattern. In good agreement, TAA1, a key gene in auxin biosynthesis, had lower expression after loss of rooting capacity. The same applied to the auxin receptor gene TIR1, suggesting reduced auxin sensitivity. On the other hand, genes associated with auxin response repression (TPL, IAA12) or with the action of cytokinins, the rhizogenesis inhibitor-related ARR1, showed higher expression in plants with lower rooting competence. Taken together, data suggest that age negatively affects E. globulus rooting by a combination of factors. Decreased endogenous auxin concentration, possibly caused by less biosynthesis, lower auxin sensitivity, higher expression of genes inhibiting auxin action, as well as of genes related to the action of cytokinins, appear to play roles in this process.

Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

Purification of an Immunoadjuvant Saponin Fraction from Quillaja brasiliensis Leaves by Reversed-Phase Silica Gel Chromatography.

Saponins include a large variety of molecules that find several applications in pharmacology. The use of Quillaja saponaria saponins as immunological adjuvants in vaccines is of interest due to their capacity to stimulate both humoral and cellular responses. The congener species Q. brasiliensis has saponins with chemical similarities and adjuvant activity comparable to that of Q. saponaria fraction Quil-A®, with additional advantages of showing lower toxicity and reduced hemolytic activity. Here we describe in detail the methods for preparing the aqueous extract from Q. brasiliensis leaves, as well as the purification of the bioactive saponin fraction QB-90 using silica reversed-phase chromatography.

Isolation and Characterization of Cyclotides from Brazilian Psychotria: Significance in Plant Defense and Co-occurrence with Antioxidant Alkaloids.

Plants from the genus Psychotria include species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata.

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis.

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta ß-pinene synthase [PcTPS-(-)ß-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

A rabies vaccine adjuvanted with saponins from leaves of the soap tree (Quillaja brasiliensis) induces specific immune responses and protects against lethal challenge.

Quillaja brasiliensis (Quillajaceae) is a saponin producing species native from southern Brazil and Uruguay. Its saponins are remarkably similar to those of Q. saponaria, which provides most of the saponins used as immunoadjuvants in vaccines. The immunostimulating capacities of aqueous extract (AE) and purified saponin fraction (QB-90) obtained from leaves of Q. brasiliensis were favorably comparable to those of a commercial saponin-based adjuvant preparation (Quil-A) in experimental vaccines against bovine herpesvirus type 1 and 5, poliovirus and bovine viral diarrhea virus in mice model. Herein, the immunogenicity and protection efficacy of rabies vaccines adjuvanted with Q. brasiliensis AE and its saponin fractions were compared with vaccines adjuvanted with either commercial Quil-A or Alum. Mice were vaccinated with one or two doses (on days 0 and 14) of one of the different vaccines and serum levels of total IgG, IgG1 and IgG2a were quantified over time. A challenge experiment with a lethal dose of rabies virus was carried out with the formulations. Viral RNA detection in the brain of mice was performed by qPCR, and RNA copy-numbers were quantified using a standard curve of in vitro transcribed RNA. All Q. brasiliensis saponin-adjuvanted vaccines significantly enhanced levels of specific IgG isotypes when compared with the no adjuvant group (P ≤ 0.05). Overall, one or two doses of saponin-based vaccine were efficient to protect against the lethal rabies exposure. Both AE and saponin fractions from Q. brasiliensis leaves proved potent immunological adjuvants in vaccines against a lethal challenge with a major livestock pathogen, hence confirming their value as competitive or complementary sustainable alternatives to saponins of Q. saponaria.

Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction.

Tapping technique comprises the extraction of pine oleoresin, a non-wood forest product consisting of a complex mixture of mono, sesqui, and diterpenes biosynthesized and exuded as a defense response to wounding. Oleoresin is used to produce gum rosin, turpentine, and their multiple derivatives. Oleoresin yield and quality are objects of interest in pine tree biotechnology, both in terms of environmental and genetic control. Monitoring these parameters in individual trees grown in the field provides a means to examine the control of terpene production in resin canals, as well as the identification of genetic-based differences in resinosis. A typical method of tapping involves the removal of bark and application of a chemical stimulant on the wounded area. Here we describe the methods for preparing the resin-stimulant paste with different adjuvants, as well as the bark streaking process in adult pine trees.

A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Alternative inactivated poliovirus vaccines adjuvanted with Quillaja brasiliensis or Quil-a saponins are equally effective in inducing specific immune responses.

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.

Shoot accumulation kinetics and effects on herbivores of the wound-induced antioxidant indole alkaloid brachycerine of Psychotria brachyceras.

A major shoot-specific monoterpene indole alkaloid produced by Psychotria brachyceras, brachycerine, is regulated by either wounding or jasmonate application. Highest concentrations of the alkaloid are found in inflorescences, suggesting a defence role. Brachycerine has antimutagenic and antioxidant properties, capable of quenching singlet oxygen, hydroxyl radical, and superoxide. This study aimed at characterizing the putative role of brachycerine in P. brachyceras responses to wounding and herbivory. Damage to leaves increased the content of brachycerine locally. Wounding did not affect phenolics content in P brachyceras leaves, and no tannins were detected in the species. In generalist herbivore bioassays, neither brachycerine nor P. brachyceras extracts showed toxic effects. In vivo hydrogen peroxide staining assay showed less wound-generated peroxide accumulation in alkaloid treated tissues. This pattern was confirmed in quantitative assays measuring tissue hydrogen peroxide concentrations. Data indicate that brachycerine is not a herbivore deterrent, but rather an indirect chemical defence, modulating oxidative stress caused by mechanical damage.

When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings.

Adventitious rooting (AR) is a multifactorial response leading to new roots at the base of stem cuttings, and the establishment of a complete and autonomous plant. AR has two main phases: (a) induction, with a requirement for higher auxin concentration; (b) formation, inhibited by high auxin and in which anatomical changes take place. The first stages of this process in severed organs necessarily include wounding and water stress responses which may trigger hormonal changes that contribute to reprogram target cells that are competent to respond to rooting stimuli. At severance, the roles of jasmonate and abscisic acid are critical for wound response and perhaps sink strength establishment, although their negative roles on the cell cycle may inhibit root induction. Strigolactones may also inhibit AR. A reduced concentration of cytokinins in cuttings results from the separation of the root system, whose tips are a relevant source of these root induction inhibitors. The combined increased accumulation of basipetally transported auxins from the shoot apex at the cutting base is often sufficient for AR in easy-to-root species. The role of peroxidases and phenolic compounds in auxin catabolism may be critical at these early stages right after wounding. The events leading to AR strongly depend on mother plant nutritional status, both in terms of minerals and carbohydrates, as well as on sink establishment at cutting bases. Auxins play a central role in AR. Auxin transporters control auxin canalization to target cells. There, auxins act primarily through selective proteolysis and cell wall loosening, via their receptor proteins TIR1 (transport inhibitor response 1) and ABP1 (Auxin-Binding Protein 1). A complex microRNA circuitry is involved in the control of auxin response factors essential for gene expression in AR. After root establishment, new hormonal controls take place, with auxins being required at lower concentrations for root meristem maintenance and cytokinins needed for root tissue differentiation.

Determination of new immunoadjuvant saponin named QB-90, and analysis of its organ-specific distribution in Quillaja brasiliensis by HPLC.

An LC method was developed and validated in order to quantify the saponin purified fraction named QB-90 obtained from the aqueous extracts of Quillaja brasiliensis leaves. Previously, QB-90 was shown to be active as adjuvant in an experimental vaccine for herpesvirus bovine type 1 in mice. The analysis was performed using an RP-8 column with acetonitrile:water isocratic elution at 214 nm. The standard curve for QB-90 was linear over the range of 0.8-10 µg mL(-1) (r = 0.9996). The aqueous extract showed linear response in the range of 50-1000 µg mL(-1) (r = 0.9996). The proposed method showed adequate repeatability and intermediary precision. The limits of detection (LOD) and quantification (LOQ) were 0.074 and 0.248 µg mL(-1), respectively. This method was applied successfully to analyse QB-90 organ-specific distribution in field stands of Q. brasiliensis and in laboratory-grown seedlings. Leaves from young plants accumulated higher QB-90 amounts than leaves from adult trees.

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

BACKGROUND: Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. RESULTS: By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs identified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. CONCLUSION: Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

Production of plant bioactive triterpenoid saponins: elicitation strategies and target genes to improve yields.

Triterpenoid saponins are a class of plant secondary metabolites with structure derived from the precursor oxidosqualene in which one or more sugar residues are added. They have a wide range of pharmacological applications, such as antiplatelet, hypocholesterolemic, antitumoral, anti-HIV, immunoadjuvant, anti-inflammatory, antibacterial, insecticide, fungicide and anti-leishmanial agents. Their accumulation in plant cells is stimulated in response to changes mediated by biotic and abiotic elicitors. The enhancement of saponin yields by methyl jasmonate in plants and cell cultures in several species indicates the involvement of these metabolites in plant defence mechanisms. The elucidation of their biosynthesis at the molecular level has advanced recently. Most studies to date have focused on the participation of early enzymes in the pathway, including oxidosqualene cyclase, squalene synthase and dammarenediol synthase, as well as in isolating and characterizing genes that encode beta-amyrin synthase. Yields of bioactive saponins in various plant species and experimental systems have been successfully increased by treating cells and tissues with jasmonate or by exposing these to oxidative stress. These elicitation and molecular studies are consolidating a robust knowledge platform from which to launch the development of improved sources for commercial supply of bioactive saponins.

Mineral nutrition and adventitious rooting in microcuttings of Eucalyptus globulus.

We characterized the adventitious rooting response of Eucalyptus globulus Labill. to various concentrations of calcium, nitrogen, phosphorus, iron, manganese, zinc, boron and copper. The parameters analyzed were percent rooting, root number, root length and mean rooting time. Root number and root length were significantly affected by mineral nutrition, whereas mean rooting time and rooting percentage seemed to be closely related to auxin availability. Root number was affected by calcium, nitrogen source and zinc, whereas root length was influenced by concentrations of phosphorus, iron and manganese, and by nitrogen source. Based on these results, we evaluated various combinations of several concentrations of these minerals in each rooting phase. Cuttings that were rooted in an optimized mineral nutrient medium and acclimatized to ex-vitro conditions for two months showed significantly higher survival after transplanting and drought stress than cuttings rooted in basal medium and treated in the same way.