Improving water deficit tolerance of Salvia officinalis L. using putrescine.

To study the effects of foliar application of putrescine (distilled water (0), 0.75, 1.5, and 2.25 mM) and water deficit stress (20%, 40%, 60%, and 80% available soil water depletion (ASWD)) on the physiological, biochemical, and molecular attributes of Salvia officinalis L., a factorial experiment was performed in a completely randomized design with three replications in the growth chamber. The results of Real-Time quantitative polymerase chain reaction (qRT-PCR) analysis showed that putrescine concentration, irrigation regime, and the two-way interaction between irrigation regime and putrescine concentration significantly influenced cineole synthase (CS), sabinene synthase (SS), and bornyl diphosphate synthase (BPPS) relative expression. The highest concentration of 1,8-cineole, camphor, α-thujone, ß-thujone, CS, SS, and BPPS were obtained in the irrigation regime of 80% ASWD with the application of 0.75 mM putrescine. There was high correlation between expression levels of the main monoterpenes synthase and the concentration of main monoterpenes. The observed correlation between the two enzyme activities of ascorbate peroxidase (APX) and catalase (CAT) strongly suggests they have coordinated action. On the other hand, the highest peroxidase (PO) and superoxide dismutase (SOD) concentrations were obtained with the application of 0.75 mM putrescine under the irrigation regime of 40% ASWD. Putrescine showed a significant increase in LAI and RWC under water deficit stress. There was an increasing trend in endogenous putrescine when putrescine concentration was increased in all irrigation regimes. Overall, the results suggest that putrescine may act directly as a stress-protecting compound and reduced H2O2 to moderate the capacity of the antioxidative system, maintain the membrane stability, and increase secondary metabolites under water deficit stress.

Sequential Response of Sage Antioxidant Metabolism to Chilling Treatment.

Chilling influences the growth and metabolism of plants. The physiological response and acclimatization of genotypes in relation to stress stimulus can be different. Two sage cultivars: 'Icterina' and 'Purpurascens' were subjected to 4 °C and 18 °C (control), and sampled between the 5th and 14th day of the treatment. Ascorbate peroxidase (APX) activity was up-regulated in chilled 'Purpurascens' on the 14th day, while guaiacol peroxidase (GPX) activity increased on the 10th and 12th day in relation to the control. GPX activity of the control 'Icterina' was frequently higher than chilled plants, and chilling did not affect APX activity of that cultivar. Catalase activity remained stable in both sage cultivars. Chilled 'Purpurascens' showed a significant increase in total phenolics contents on the 5th, 7th, and 12th day and in total antioxidant capacity on the 5th and 10th day as compared to the control for respective sampling days. Higher malondialdehyde content was found in chilled plants on the 12th, or 14th day, differences reached 26-28% of the controls. Chilling caused significant decrease in dry matter content. The stress response was more stable and effective in 'Icterina', while more dynamic changes were found for 'Purpurascens'. Based on our results, we propose to use 'Purpurascens' for targeted stress-induced studies and 'Icterina' for field applications.

Signalling molecules responsive to ozone-induced oxidative stress in Salvia officinalis.

Tropospheric ozone (O3) is the most important gaseous pollutant and induces a mass of negative impacts on vegetation at functional and genic levels. The aim of the present study was to investigate the role of reactive oxygen species and signalling molecules in sage plants exposed to O3 (200 ppb, 5 h). Ozone exposure induced only a transient oxidative burst, as confirmed by the rapid peak of anion superoxide during the first hours of exposure (+16% compared to controls). The spontaneous reaction of O3 with membrane fatty acids stimulates peroxidative processes, as demonstrated by the rise of thiobarbituric acid reactive substances concentration starting after 1 h of exposure (+25%). The formation of lipid-based signalling molecules (e.g. jasmonic acid) may be regarded as a sort of O3-perception. The concomitant accumulation of salicylic acid suggests that sage responds early to O3 by inducing cellular antioxidants mechanisms in order to minimize O3-oxidative burst. The transient increase of abscisic acid (+25% at the end of the treatment) twinned with the maximal ethylene emission (about two-fold higher than controls) could be interpreted as a first attempt by plants to regulate the signalling responses induced by O3. In order to investigate the involvement of transcription factors in managing oxidative protection, BLASTX analysis against the Salvia miltiorrhiza sequence genome was carried out using Arabidopsis thaliana WRKY sequences as queries. Six gene sequences were identified for sage WRKYs and their relative gene expression analyses were characterized. WRKY4, WRKY5, WRKY11 and WRKY46 were up-regulated by O3 at 2 and 5 h of exposure and they showed similarity with AtWRKY48, AtWRKY22 and AtWRKY53 in A. thaliana. These results suggest that WRKYs could play a pivotal role in the signalling mechanisms during the responses of plants to O3.

Probability of lek collapse is lower inside sage-grouse Core Areas: Effectiveness of conservation policy for a landscape species.

Greater sage-grouse (Centrocercus urophasianus) occupy sagebrush (Artemisia spp.) habitats in 11 western states and 2 Canadian provinces. In September 2015, the U.S. Fish and Wildlife Service announced the listing status for sage-grouse had changed from warranted but precluded to not warranted. The primary reason cited for this change of status was that the enactment of new regulatory mechanisms was sufficient to protect sage-grouse populations. One such plan is the 2008, Wyoming Sage Grouse Executive Order (SGEO), enacted by Governor Freudenthal. The SGEO identifies "Core Areas" that are to be protected by keeping them relatively free from further energy development and limiting other forms of anthropogenic disturbances near active sage-grouse leks. Using the Wyoming Game and Fish Department's sage-grouse lek count database and the Wyoming Oil and Gas Conservation Commission database of oil and gas well locations, we investigated the effectiveness of Wyoming's Core Areas, specifically: 1) how well Core Areas encompass the distribution of sage-grouse in Wyoming, 2) whether Core Area leks have a reduced probability of lek collapse, and 3) what, if any, edge effects intensification of oil and gas development adjacent to Core Areas may be having on Core Area populations. Core Areas contained 77% of male sage-grouse attending leks and 64% of active leks. Using Bayesian binomial probability analysis, we found an average 10.9% probability of lek collapse in Core Areas and an average 20.4% probability of lek collapse outside Core Areas. Using linear regression, we found development density outside Core Areas was related to the probability of lek collapse inside Core Areas. Specifically, probability of collapse among leks >4.83 km from inside Core Area boundaries was significantly related to well density within 1.61 km (1-mi) and 4.83 km (3-mi) outside of Core Area boundaries. Collectively, these data suggest that the Wyoming Core Area Strategy has benefited sage-grouse and sage-grouse habitat conservation; however, additional guidelines limiting development densities adjacent to Core Areas may be necessary to effectively protect Core Area populations.

Diel variations in carbon isotopic composition and concentration of organic acids and their impact on plant dark respiration in different species.

Leaf respiration in the dark and its C isotopic composition (δ(13) CR ) contain information about internal metabolic processes and respiratory substrates. δ(13) CR is known to be less negative compared to potential respiratory substrates, in particular shortly after darkening during light enhanced dark respiration (LEDR). This phenomenon might be driven by respiration of accumulated (13) C-enriched organic acids, however, studies simultaneously measuring δ(13) CR during LEDR and potential respiratory substrates are rare. We determined δ(13) CR and respiration rates (R) during LEDR, as well as δ(13) C and concentrations of potential respiratory substrates using compound-specific isotope analyses. The measurements were conducted throughout the diel cycle in several plant species under different environmental conditions. δ(13) CR and R patterns during LEDR were strongly species-specific and showed an initial peak, which was followed by a progressive decrease in both values. The species-specific differences in δ(13) CR and R during LEDR may be partially explained by the isotopic composition of organic acids (e.g., oxalate, isocitrate, quinate, shikimate, malate), which were (13) C-enriched compared to other respiratory substrates (e.g., sugars and amino acids). However, the diel variations in both δ(13) C and concentrations of the organic acids were generally low. Thus, additional factors such as the heterogeneous isotope distribution in organic acids and the relative contribution of the organic acids to respiration are required to explain the strong (13) C enrichment in leaf dark-respired CO2 .

Expression Analysis of Phenylalanine Ammonia Lyase Gene and Rosmarinic Acid Production in Salvia officinalis and Salvia virgata Shoots Under Salicylic Acid Elicitation.

Partial fragments of phenylalanine ammonia lyase (PAL) genes were cloned and characterized from Salvia officinalis (SoPAL) and Salvia virgata (SvPAL). Different concentrations (250 and 500 µM) of exogenous salicylic acid (SA) were used when correlation between PAL expression and rosmarinic acid (RA) accumulation was compared. The results showed that the deduced cDNA sequences of the partial genes had high similarities with those of known PAL gene from other plant species. Semi-quantitative reverse transcription PCR (RT-PCR) analysis revealed that exogenous application of SA led to up-regulating of the PAL expression. Further analysis showed that in S. virgata, at higher concentration of SA, higher accumulation of RA was achieved, while in S. officinalis, the higher RA accumulation was observed at lower concentration of SA. It was concluded that there was no positive correlation between the intensity of PAL transcription and the RA accumulation in the studied species. Therefore, despite of the increase in transcription rate of the PAL at the higher concentration of SA, the lower amounts of RA were accumulated in the case of S. officinalis. Consequently, the hypothesis that PAL is the rate-determining step in RA biosynthesis is not always valid and probably some other unknown factors participate in the synthesis of phenolics.

Altered water and nitrogen input shifts succession in a southern California coastal sage community.

Vegetation-type conversions between grasslands and shrublands have occurred worldwide in semiarid regions over the last 150 years. Areas once covered by drought-deciduous shrubs in Southern California (coastal sage scrub) are converting to grasslands dominated by nonnative species. Increasing fire frequency, drought, and nitrogen deposition have all been hypothesized as causes of this conversion, though there is little direct evidence. We constructed rain-out shelters in a coastal sage scrub community following a wildfire, manipulated water and nitrogen input in a split-plot design, and collected annual data on community composition for four years. While shrub cover increased through time in all plots during the postfire succession, both drought and nitrogen significantly slowed recovery. Four years after the fire, average native shrub cover ranged from over 80% in water addition, ambient-nitrogen plots to 20% in water reduction, nitrogen addition plots. Nonnative grass cover was high following the fire and remained high in the water reduction plots through the third spring after the fire, before decreasing in the fourth year of the study. Adding nitrogen decreased the cover of native plants and increased the cover of nonnative grasses, but also increased the growth of one crown-sprouting shrub species. Our results suggest that extreme drought during postfire succession may slow or alter succession, possibly facilitating vegetation-type conversion of coastal sage scrub to grassland. Nitrogen addition slowed succession and, when combined with drought, significantly decreased native cover and increased grass cover. Fire, drought, and atmospheric N deposition are widespread aspects of environmental change that occur simultaneously in this system. Our results imply these drivers of change may reinforce each other, leading to a continued decline of native shrubs and conversion to annual grassland.

Effects of PAR and UV-B radiation on herbal yield, bioactive compounds and their antioxidant capacity of some medicinal plants under controlled environmental conditions.

Photosynthetically active radiation (PAR) and Ultraviolet B (UV-B) radiation are among the main environmental factors acting on herbal yield and biosynthesis of bioactive compounds in medicinal plants. The objective of this study was to evaluate the influence of biologically effective UV-B light (280-315 nm) and PAR (400-700 nm) on herbal yield, content and composition, as well as antioxidant capacity of essential oils and polyphenols of lemon catmint (Nepeta cataria L. f. citriodora), lemon balm (Melissa officinalis L.) and sage (Salvia officinalis L.) under controlled greenhouse cultivation. Intensive UV-B radiation (2.5 kJ m(-2)  d(-1) ) influenced positively the herbal yield. The essential oil content and composition of studied herbs were mainly affected by PAR and UV-B radiation. In general, additional low-dose UV-B radiation (1 kJ m(-2) d(-1) ) was most effective for biosynthesis of polyphenols in herbs. Analysis of major polyphenolic compounds provided differences in sensitivity of main polyphenols to PAR and UV-B radiation. Essential oils and polyphenol-rich extracts of radiated herbs showed essential differences in antioxidant capacity by the ABTS system. Information from this study can be useful for herbal biomass and secondary metabolite production with superior quality under controlled environment conditions.

Effects of foliar fertilization and arbuscular mycorrhizal colonization on Salvia officinalis L. growth, antioxidant capacity, and essential oil composition.

BACKGROUND: The effect of foliar fertilization and Glomus intraradices inoculation on the growth, qualitative and quantitative pattern of essential oil in Salvia officinalis was determined. Sage plants were grown in a glass house on a soil/sand mixture (w/w = 3:1). Agroleaf total, N:P:K = 20:20:20 + microelements, was used at the whole vegetative growth stage as a 0.3% solution. Inoculation with Glomus intraradices was done at the sowing stage. RESULTS: Application of foliar fertilization and/or mycorrhizal colonization improved dry biomass accumulation and increased the content of antioxidant metabolites (ascorbate and reduced glutathione). Applied treatments lowered the activities of the antioxidants enzymes catalase, ascorbate peroxidase and superoxide dismutase, while guaiacol peroxidase increased. The relative quantity of essential oil pattern was also altered as a result of the applied treatments. Combined application (FF + Gi) significantly promoted 1,8-cineole and alpha-thujone, mycorrhizal colonization enhanced bornyl acetate, 1,8-cineole, alpha- and beta-thujones, while foliar fertilization increased bornyl acetate and camphor. The favorable effect of root colonization by Glomus intraradices was determined both on quantitative and qualitative pattern of sage essential oil. CONCLUSION: We conclude that inoculation with Glomus intraradices resulted in improved essential oil yield and quality, while combined application of foliar fertilizer and mycorrhizal fungi predominantly enhanced shoot biomass accumulation.

[Study on characteristics of seed germination of Salvia officinalis].

OBJECTIVE: To investigate the influences of lightness and temperature on seeds germination of Salvia officinalis, and offer the basis for the standardized cultivation of S. officinalis. METHOD: The morphological characters, 1 000-grains weight and rate of water absorption of the seed were observed, the germination capacity, percent of germination power and germination index at the five different degrees: 10, 15, 20, 25, 30 degrees C were also measared. RESULT: 1 000-grains weight of the seeds was 8.03 g, the rate of water absorption is 49% within 24 h, the highest rate of germination capacity reached 85.33%, the germination index is 45.56. CONCLUSION: The lightness and 25 degrees C are the most suitable parameters for the seed germination of S. officinalis.

Biomass segregation in sage cell suspension culture.

The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggregates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g(-1) DW which was up to 50 times higher compared to aggregated biomass fractions.