Compensatory phenolic induction dynamics in aspen after aphid infestation.

Condensed tannins (CTs) are polyphenolics and part of the total phenolic (TP) pool that shape resistance in aspen (Populus tremula). CTs are negatively associated with pathogens, but their resistance properties against herbivores are less understood. CTs shape resistance to pathogens and chewing herbivores and could also shape resistance to aphids. Being chemical pools that are highly variable it can further be questioned whether CT-shaped resistance is better described by constitutive levels, by the induced response potential, or by both. Here, aspen genotypes were propagated and selected to represent a range of inherent abilities to produce and store foliar CTs; the plantlets were then exposed to Chaitophorus aphid infestation and to mechanical (leaf rupture) damage, and the relative abundance of constitutive and induced CTs was related to aphid fitness parameters. As expected, aphid fecundity was negatively related to CT-concentrations of the aphid infested plants although more consistently related to TPs. While TPs increased in response to damage, CT induction was generally low and it even dropped below constitutive levels in more CT-rich genotypes, suggesting that constitutive CTs are more relevant measurements of resistance compared to induced CT-levels. Relating CT and TP dynamics with phenolic low molecular compounds further suggested that catechin (the building block of CTs) increased in response to aphid damage in amounts that correlated negatively with CT-induction and positively with constitutive CT-levels and aphid fecundity. Our study portrays dynamic phenolic responses to two kinds of damage detailed for major phenylpropanoid classes and suggests that the ability of a genotype to produce and store CTs may be a measurement of resistance, caused by other, more reactive, phenolic compounds such as catechin. Rupture damage however appeared to induce catechin levels oppositely supporting that CTs may respond differently to different kinds of damage.

A metabolomic study of Gomphrena agrestis in Brazilian Cerrado suggests drought-adaptive strategies on metabolism.

Drought is the main factor that limits the distribution and productivity of plant species. In the Brazilian Cerrado, the vegetation is adapted to a seasonal climate with long- and short-term periods of drought. To analyze the metabolic strategies under such conditions, a metabolomic approach was used to characterize Gomphrena agrestis Mart. (Amaranthaceae) a native species that grows under natural conditions, in a rock-field area. Roots and leaves material from native specimens were sampled along different seasons of the year and LC-MS and GC-MS analyzed for multiple chemical constituents. The datasets derived from the different measurements were combined and evaluated using multivariate analysis. Principal component analysis was used to obtain an overview of the samples and identify outliers. Later, the data was analyzed with orthogonal projection to latent structures discriminant analysis to obtain valid models that could explain the metabolite variations in the different seasons. Two hundred and eighty metabolites were annotated, generating a unique database to characterize metabolic strategies used to cope with the effects of drought. The accumulation of fructans in the thickened roots is consistent with the storage of carbons during the rainy season to support the energy demand during a long period of drought. The accumulation of Abscisic acid, sugars and sugar alcohols, phenolics, and pigment in the leaves suggests physiological adaptations. To cope with long-term drought, the data suggests that tissue water status and storage of reserves are important to support plant survival and regrowth. However, during short-term drought, osmoregulation and oxidative protection seems to be essential, probably to support the maintenance of active photosynthesis.

Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

No evidence of geographical structure of salicinoid chemotypes within Populus tremula.

Salicinoids are well-known defense compounds in salicaceous trees and careful screening at the population level is warranted to fully understand their diversity and function. European aspen, Populus tremula, is a foundation species in Eurasia and highly polymorphic in Sweden. We exhaustively surveyed 102 replicated genotypes from the Swedish Aspen collection (SwAsp) for foliar salicinoids using UHPLC-ESI-TOF/MS and identified nine novel compounds, bringing the total to 19 for this species. Salicinoid structure followed a modular architecture of a salicin skeleton with added side groups, alone or in combination. Two main moieties, 2'-cinnamoyl and 2'-acetyl, grouped the SwAsp population into four distinct chemotypes, and the relative allocation of salicinoids was remarkably constant between different environments, implying a highly channeled biosynthesis of these compounds. Slightly more than half of the SwAsp genotypes belonged to the cinnamoyl chemotype. A fraction synthesized the acetyl moiety alone (∼7%) or in combination with cinnamoyl (∼2%), and close to forty percent lacked either of the two characteristic moieties, and thus resemble P. tremuloides in their salicinoid profile. The two most abundant chemotypes were evenly distributed throughout Sweden, unlike geographical patterns reported for SwAsp phenology traits, plant defense genes, and herbivore community associations. Here we present the salicinoid characterization of the SwAsp collection as a resource for future studies of aspen chemical ecology, salicinoid biosynthesis, and genetics.

UHPLC-ESI/TOFMS determination of salicylate-like phenolic gycosides in Populus tremula leaves.

Associations of salicylate-like phenolic glycosides (PGs) with biological activity have been reported in Salix and Populus trees, but only for a few compounds, and in relation to a limited number of herbivores. By considering the full diversity of PGs, we may improve our ability to recognize genotypes or chemotype groups and enhance our understanding of their ecological function. Here, we present a fast and efficient general method for salicylate determination in leaves of Eurasian aspen that uses ultra-high performance liquid chromatography-electrospray ionization/time-of-flight mass spectrometry (UHPLC-ESI/TOFMS). The time required for the liquid chromatography separations was 13.5 min per sample, compared to around 60 min per sample for most HPLC protocols. In leaf samples from identical P. tremula genotypes with diverse propagation and treatment histories, we identified nine PGs. We found the compound-specific mass chromatograms to be more informative than the UV-visible chromatograms for compound identification and when quantitating samples with large variability in PG content. Signature compounds previously reported for P. tremoloides (tremulacin, tremuloidin, salicin, and salicortin) always were present, and five PGs (2'-O-cinnamoyl-salicortin, 2'-O-acetyl-salicortin, 2'-O-acetyl-salicin, acetyl-tremulacin, and salicyloyl-salicin) were detected for the first time in P. tremula. By using information about the formic acid adduct that appeared for PGs in the LTQ-Orbitrap MS environment, novel compounds like acetyl-tremulacin could be tentatively identified without the use of standards. The novel PGs were consistently either present in genotypes regardless of propagation and damage treatment or were not detectable. In some genotypes, concentrations of 2'-O-acetyl-salicortin and 2'-O-cinnamoyl-salicortin were similar to levels of biologically active PGs in other Salicaceous trees. Our study suggests that we may expect a wide variation in PG content in aspen populations which is of interest both for studies of interactions with herbivores and for mapping population structure.

Indução de metabólitos secundários em plântulas de Hypericum brasiliense Choisy crescendo in vitro / Induction of secondary metabolites in plantlets of Hypericum brasiliense Choisy in vitro

A produção de rutina, quercetina, 1,5-diidroxixantona e ácido betulínico foi investigada em plântulas de H. brasiliense crescendo in vitro, sob a influência de ácido salicílico, polietilenoglicol, NaCl, 24-epibrassinolídeo, benzotiadiazole (BION), metiljasmonato e concentrações aumentadas de boro e nitrogênio no meio líquido de cultura. As avaliações foram feitas após 5 e 10 dias do início dos tratamentos. Os maiores aumentos de conteúdo foram observados com quercetina para boro e ácido salicílico aos 5 dias, e 24-epibrassinolídeo e BION aos 10 dias.

The production of rutin, quercetin, 1,5-dihydroxyxanthone and betulinic acid was investigated in plantlets of H. brasieliense in vitro, and exposed to salycilic acid, poliethylene glycol, NaCl, 24-epibrassinolide, benzothiadiazole (BION), methyljasmonate and increased concentrations of boron and nitrogen in the liquid culture medium. Evaluations of the contents were carried out after 5 and 10 days of treatments. The highest increase was observed in quercetin in the salycilic acid and B treatments after 5 days of exposure, and in 24-epibrassinolide and BION after 10 days.

HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus.

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.