Modified distribution in the polyphenolic profile of rosemary leaves induced by plant inoculation with an arbuscular mycorrhizal fungus.

BACKGROUND: Rosemary forms an arbuscular mycorrhizal (AM) symbiosis with a group of soilborne fungi belonging to the phylum Glomeromycota, which can modify the plant metabolome responsible for the antioxidant capacity and other health beneficial properties of rosemary. RESULTS: The effect of inoculating rosemary plants with an AM fungus on their growth via their polyphenolic fingerprinting was evaluated after analyzing leaf extracts from non-inoculated and inoculated rosemary plants by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Plant growth parameters indicated that mycorrhizal inoculation significantly increased plant height and biomass. Chemical modifications in the plant polyphenolic profile distribution were found after a principal components analysis (PCA) loading plots study. Four compounds hosting strong antioxidant properties - ferulic acid, asiatic acid, carnosol, and vanillin - were related to mycorrhizal rosemary plants while caffeic and chlorogenic acids had a higher influence on non-mycorrhizal plants. CONCLUSION: Mycorrhization was found to stimulate growth to obtain a higher biomass of plant leaves in a short time, avoiding chemical fertilization, while analytical results demonstrate that there is an alteration in the distribution of polyphenols in plants colonized by the symbiotic fungus, which can be related to an improvement in nutritional properties with future industrial significance. © 2018 Society of Chemical Industry.

Species-specific differences in temporal and spatial variation in δ(13)C of plant carbon pools and dark-respired CO (2) under changing environmental conditions.

Stable carbon isotope signatures are often used as tracers for environmentally driven changes in photosynthetic δ(13)C discrimination. However, carbon isotope signatures downstream from carboxylation by Rubisco are altered within metabolic pathways, transport and respiratory processes, leading to differences in δ(13)C between carbon pools along the plant axis and in respired CO(2). Little is known about the within-plant variation in δ(13)C under different environmental conditions or between species. We analyzed spatial, diurnal, and environmental variations in δ(13)C of water soluble organic matter (δ(13)C(WSOM)) of leaves, phloem and roots, as well as dark-respired δ(13)CO(2) (δ(13)C(res)) in leaves and roots. We selected distinct light environments (forest understory and an open area), seasons (Mediterranean spring and summer drought) and three functionally distinct understory species (two native shrubs-Halimium halimifolium and Rosmarinus officinalis-and a woody invader-Acacia longifolia). Spatial patterns in δ(13)C(WSOM) along the plant vertical axis and between respired δ(13)CO(2) and its putative substrate were clearly species specific and the most δ(13)C-enriched and depleted values were found in δ(13)C of leaf dark-respired CO(2) and phloem sugars, ~-15 and ~-33 ‰, respectively. Comparisons between study sites and seasons revealed that spatial and diurnal patterns were influenced by environmental conditions. Within a species, phloem δ(13)C(WSOM) and δ(13)C(res) varied by up to 4 ‰ between seasons and sites. Thus, careful characterization of the magnitude and environmental dependence of apparent post-carboxylation fractionation is needed when using δ(13)C signatures to trace changes in photosynthetic discrimination.

Comprehensive study of honey with protected denomination of origin and contribution to the enhancement of legal specifications.

In this study the characterization of a total of 60 honey samples with Protected Denomination of Origin (PDO) collected over three harvests (2009-2011, inclusive), from the Northeast of Portugal was carried out based on the presence of pollen, physicochemical and microbiological characteristics. All samples were found to meet the European Legislation, but some didn't meet the requirements of the PDO specifications. Concerning the floral origin of honey, our results showed the prevalence of rosemary (Lavandula pedunculata) pollen. The microbiological quality of all the analyzed samples was satisfactory, since fecal coliforms, sulfite-reducing clostridia and Salmonella were absent, and molds and yeasts were detected in low counts. Significant differences between the results were studied using one-way analysis of variance (ANOVA), followed by Tukey's HSD test. The samples were submitted to discriminant function analysis, in order to determine which variables differentiate between two or more naturally occurring groups (Forward Stepwise Analysis). The variables selected were in this order: diastase activity, pH, reducing sugars, free acidity and HMF. The pollen spectrum has perfect discriminatory power. This is the first study in which a honey with PDO was tested, in order to assess its compliance with the PDO book of specifications.

Inhibition of anticancer drug efflux transporter P-glycoprotein by rosemary phytochemicals.

The effects of dietary antioxidative and chemopreventive rosemary phytochemicals on the function of the human drug efflux transporter P-glycoprotein (MDR1, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytochemicals found in rosemary such as carnosic acid, carnosol, rosmarinic acid, and ursolic acid were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB-C2 cells increased in the presence of carnosic acid, carnosol, and ursolic acid in a concentration-dependent manner. In contrast, carnosic acid, carnosol, rosmarinic acid, and ursolic acid had no effects on the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-glycoprotein were stimulated by carnosic acid, carnosol, and ursolic acid. KB-C2 cells were sensitized to vinblastine cytotoxicity by carnosic acid, showing that carnosic acid reverses multidrug resistance. These results suggest that rosemary phytochemicals, such as carnosic acid, have inhibitory effects on anticancer drug efflux transporter P-glycoprotein and may become useful to enhance the efficacy of cancer chemotherapy.

Dried leaves of Rosmarinus officinalis as a treatment for streptococcosis in tilapia.

Dietary application of dried Rosmarinus officinalis (rosemary) leaves as a treatment for streptococcal infection was studied in tilapia, Oreochromis sp. Feeding with dried rosemary leaves significantly reduced mortality following infection with Streptococcus iniae: 44% mortality in the group fed 8% rosemary, similar to oxytetracycline treatment (43% mortality), and significantly lower than the control (65%). Dietary administration of 16% rosemary significantly reduced mortality because of Streptococcus agalactiae infection in 44 g fish (62% and 76% in 16% rosemary and control, respectively), but not in a similar experiment conducted with 5.5 g fish. The antibacterial effect of rosemary on S. iniae was studied. Activity of rosemary cultivar Israel was reduced during the winter, but there was no significant change in cultivars Oranit and Star. Storage of powdered rosemary leaves at 50 degrees C resulted in fourfold and eightfold higher MIC(24 h) values after 3 and 4.5 months, respectively. Storage at -20 degrees C, 4 degrees C and 25 degrees C and autoclaving (120 degrees C) each resulted in a twofold increase in MIC(24 h). Repeated exposures of S. iniae to rosemary did not affect minimal inhibitory concentration, suggesting no development of resistance to rosemary.

Implication of phytometabolites on metal tolerance of the pseudo-metallophyte -Rosmarinus officinalis- in a Mediterranean brownfield.

This study highlights the trace metal and metalloid (TMM) accumulation in Rosmarinus officinalis L. and its chemical responses when exposed to high levels of contamination. R. officinalis individuals growing along a gradient of mixed TMM soil pollution, resulting from past industrial activities, were analysed. Several plant secondary metabolites, known to be involved in plant tolerance to TMM or as a plant health indicator, were investigated. The levels of thiol compounds and phytochelatin precursors (cysteine and glutathione) in the shoots were measured in the laboratory, while a portable non-destructive instrument was used to determine the level of phenolic compounds and chlorophylls directly on site. The level of Pb, As, Sb and Zn contaminations within the soil and plants was also determined. The results highlighted a decrease of TMM translocation with increases of soil contamination. The concentration of TMM in the shoots followed the Mitscherlich equation and reached a plateau at 0.41, 7.9, 0.37, 51.3 mg kg-1 for As, Pb, Sb and Zn, respectively. In the shoots, the levels of thiols and phenols were correlated to concentrations of TMM. Glutathione seems to be the main thiol compounds involved in the tolerance to As, Pb and Sb. Phenols indices, using non-destructive measurements, may be considered as an easy way to establish a proxy to estimate the TMM contamination level of the R. officinalis shoots. The study highlights metabolic processes that contribute to the high potential of R. officinalis for phytostabilisation of TMM in contaminated areas in the Mediterranean.

How do Mediterranean shrub species cope with shade? Ecophysiological response to different light intensities.

Under natural conditions, light exposure for Mediterranean shrubs can be highly variable, especially during cloudy days or under a canopy, and can interfere with other environmental factors such as temperature and water availability. With the aim of decoupling the effect of radiation and temperature from water availability, we conducted an experiment where two perennial and three summer semi-deciduous shrub species were subjected to different levels of irradiation. In order to follow plant responses to light exposure, we measured gas exchange, photosystem II photochemical efficiency, photosynthetic pigments and leaf mass area in spring and summer. Results showed that all study species presented a plastic response to different light conditions, and that light-related traits varied in a coordinated manner. Summer semi-deciduous species exhibited a more opportunistic response, with higher photosynthesis rates in full sun, but under shade conditions, the two strategies presented similar assimilation rates. Stomatal conductance did not show such a drastic response as photosynthetsis, being related to changes in WUE. Daily cycles of Fv /Fm revealed a slight photoinhibitory response during summer, mainly in perennial species. In all cases photosynthetic pigments adjusted to the radiation level; leaves had lower chlorophyll content, higher pool of xanthophylls and higher proportion of the de-epoxydaded state of xanthophylls under sun conditions. Lutein content increased in relation to the xanthophyll pool under shade conditions. Our results evidenced that radiation is an important driving factor controlling morphological and physiological status of Mediterranean shrub species, independently of water availability. Summer semi-deciduous species exhibit a set of traits with higher response variability, maximising their photosynthetic assimilation under different sun conditions.

Seed germination and seedling allogamy in Rosmarinus officinalis: the costs of inbreeding.

Self-pollination by geitonogamy is likely in self-compatible plants that simultaneously expose a large number of flowers to pollinators. However, progeny of these plants is often highly allogamous. Although mechanisms to increase cross-pollination have been identified and studied, their relative importance has rarely been addressed simultaneously in plant populations. We used Rosmarinus officinalis to explore factors that influence the probability of self-fertilisation due to geitonogamy or that purge its consequences, focusing on their effects on seed germination and allogamy rate. We experimentally tested the effect of geitonogamy on the proportion of filled seeds and how it influences germination rate. During two field seasons, we studied how life history and flowering traits of individuals influence seed germination and allogamy rates of their progeny in wild populations at the extremes of the altitudinal range. The traits considered were plant size, population density, duration of the flowering season, number of open flowers, flowering synchrony among individuals within populations and proportion of male-sterile flowers. We found that most seeds obtained experimentally from self-pollination were apparently healthy but empty, and that the proportion of filled seeds drove the differences in germination rate between self- and cross-pollination experiments. Plants from wild populations consistently had low germination rate and high rate of allogamy, as determined with microsatellites. Germination rate related positively to the length of the flowering season, flowering synchrony and the ratio of male-sterile flowers, whereas the rate of allogamous seedlings was positively related only to the ratio of male-sterile flowers. Rosemary plants purge most of the inbreeding caused by its pollination system by aborting the seeds. This study showed that the rates of seed germination and allogamy of the seedlings depend on a complex combination of factors that vary in space and time. Male sterility of flowers, length of the flowering season and flowering synchrony of individuals within populations all favour high rates of cross-pollination, therefore increasing germination and allogamy rates. Flowering traits appear to be highly plastic and respond to local and seasonal conditions.

Water deficit stress induces different monoterpene and sesquiterpene emission changes in Mediterranean species. Relationship between terpene emissions and plant water potential.

The effects of water deficit stress and plant water potential (psi) on monoterpene and sesquiterpene leaf emissions from Rosmarinus officinalis, Pinus halepensis, Cistus albidus and Quercus coccifera were studied over 11 days of water withholding (from t(1) to t(11)), after substrates had achieved their field capacity (control pots: t(0)). Volatile compounds were sampled from the same twig per plant all throughout the study, using a dynamic bag enclosure system. Volatiles, collected in Tenax TA, were studied by means of GC-FID and GC-MS. Monoterpene emissions of water stressed plants (t(1)-t(11)) were either similar to those of control seedlings (R. officinalis and Q. coccifera) or higher (P. halepensis and C. albidus). By contrast, sesquiterpene emissions were strongly reduced or inhibited after four days of water withholding, particularly for R. officinalis, thus altering terpene emission composition. Despite the positive effect of water stress on leaf monoterpene emissions of P. halepensis and C. albidus, the significant correlation between these emissions and psi showed a slow decrease of these emissions over long term water deficit periods. This contrasted with the rapid decline of sesquiterpene emissions of R. officinalis according to lower values of psi. These results provide an overall picture of the different responses of monoterpene and sesquiterpene emissions to progressive water loss. They also reveal the utility of using psi for estimating some emission rates of some species according to drought conditions.

Enhanced alpha-tocopherol quinone levels and xanthophyll cycle de-epoxidation in rosemary plants exposed to water deficit during a Mediterranean winter.

Photosynthesis operates in a constantly shifting balance between efficient capture of solar energy and its rapid dissipation when captured in excess. In an attempt to better understand the role of alpha-tocopherol in plant photoprotection, we examined the changes in alpha-tocopherol quinone (alpha-TQ), in parallel with those of other low-molecular-weight antioxidants, in rosemary plants exposed to water deficit during a Mediterranean winter. Relative leaf water content (RWC) decreased from about 85% to approximately 65% in drought, but plants did not show symptoms of oxidative damage, as indicated by constant Fv/Fm ratios and malondialdehyde (MDA) levels. alpha-TQ was present at concentrations of 20 mmol per 100 mol of chlorophyll, and represented less than 1% of total tocopherol content in non-stressed leaves. Although alpha-tocopherol levels were not significantly altered, alpha-TQ reached up to 36 mmol per 100 mol of chlorophyll under stress (under both high light and after exposure to increasing water deficit at lower light intensities). Furthermore, both alpha-TQ and xanthophyll cycle de-epoxidation were strongly negatively correlated with the relative efficiency of photosystem II photochemistry (phiPSII) at midday. The biological significance of alpha-tocopherol and alpha-TQ in the network of photo- and antioxidative protection mechanisms evolved by plants to withstand stress is discussed.

Drought and soil amendment effects on monoterpene emission in rosemary plants.

The aim of this work was to study the changes during 15days in the monoterpene emission rates of the Mediterranean shrub rosemary (Rosmarinus officinalis L.), in response to increasing drought stress and fertilisation using two different composts derived from livestock anaerobic digestates (cattle and pig slurry). Drought stress considerably reduced photosynthetic rates, stomatal conductance and isoprenoid emissions and also induced a change in blend composition. In the drought stressed rosemary plants, a positive relationship of non-oxygenated monoterpene emissions and a negative relationship of oxygenated monoterpene with photosynthesis were observed, indicating a different control mechanism over the emissions of the two types of isoprenoids. The emission of non-oxygenated monoterpenes seemed to depend more on photosynthesis and "de novo" synthesis, whereas emission of oxygenate monoterpenes was more dependent on volatilisation from storage, mainly driven by cumulative temperatures. In the short term, the addition of composted organic materials to the soil did not induce a significant effect on isoprenoid emission rates in the rosemary plants. However, the effect of the interaction between fertilisation and seasonality on isoprenoid emission rates was influenced by the amendment origin. Also, we emphasized changes in potential isoprenoid emission factors throughout the experiment, probably indicating changes in the leaf developmental stage.