Stevan Avramov

Simultaneous UHPLC/DAD/(+/−)HESI–MS/MS Analysis of Phenolic Acids and Nepetalactones in Methanol Extracts of Nepeta Species: A Possible Application in Chemotaxonomic Studies

INTRODUCTION Nepeta species contain a variety of secondary metabolites, including iridoid monoterpenes - nepetalactones and phenolic acids - that are considered the main bioactive constituents. This work represents the first attempt to comparatively explore variations in these two major groups of secondary metabolites within the genus. OBJECTIVE To develop an efficient analytical methodology for simultaneous analysis of nepetalactones and phenolic acids in methanol extracts of selected Nepeta species, and to evaluate its potential application in chemotaxonomic studies. MATERIAL AND METHODS A UHPLC combined with linear-trap quadrupole (LTQ) orbitrap MS method was used to characterise chemical diversity and complexity of phenolics among 12 selected Nepeta species. A targeted metabolomic approach using UHPLC coupled to a diode array detector (DAD) and combined with (+/-) heated electrospray ionisation (HESI) MS/MS was developed and validated for quantitative analysis of six hydroxycinnamic acid derivatives and four nepetalactones. RESULTS Phenolic profiling provided a valuable database of bioactive compounds in the plant group studied, including phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and flavonoids (flavones, flavonols and flavanones). Principal component analysis and cluster analysis suggested the applicability of 10 targeted compounds as chemomarkers for chemotaxonomic studies. Pearsons correlation analysis revealed significant positive correlations between metabolites involved in different biosynthetic pathways (phenylpropanoid or monoterpenoid). CONCLUSION The described targeted metabolomic approach proved to be highly beneficial in designing a phytochemical overview of the genus Nepeta, and might have applications in further clarification of phylogenetic relations. Furthermore, it has the potential to be implemented in a routine quality control of plant material and herbal preparations.

Phenotypic plasticity in response to an irradiance gradient in Iris pumila: adaptive value and evolutionary constraints

Adaptive values of plasticity in Iris pumila leaf traits (morphological: SLA, specific leaf area; anatomical: SD, stomatal density; LT, leaf thickness; VBN, vascular bundle number; SW, sclerenchyma width; CW, cuticle width, and physiological: ChlT, total chlorophyll concentration; ChlA/B, chlorophyll a/b ratio) were tested at three irradiance levels in a growth-room. Siblings from 28 full-sib families from an open dune site and a woodland understory responded similarly to variation in light availability: SLA gradually increased, while anatomical and physiological traits decreased with light reduction. In the Dune population, standardized linear selection gradients were significant for SLA and ChlT at high light, VBN along the entire light gradient, SW at high- and low-, and ChlA/B at low-irradiance. In the Woods population, the significant standardized linear selection gradients were observed for SLA and LT at low- and VBN at both high- and low-irradiance. A significant nonlinear selection gradient was recorded for SD and LT at medium irradiance. Comparisons of the plastic responses to each light quantity with the phenotypes favored by selection in that environments revealed that only an increased SLA value at low light in the Woods population was ecologically significant (adaptive). In the Dune population, SD and VBN entailed plasticity costs at low irradiance, while a cost of homeostasis was recognized for ChlT and ChlA/B at medium light, SD and CW at high- and low-, and SLA at high- and medium-light level. In the shaded population, CW and ChlA/B incurred plasticity costs at high irradiance, while for ChlT plasticity costs appeared under medium- and low-light conditions. In all leaf traits, genetic variation for plasticity was statistically undetectable. Genetic correlations between these traits were mostly insignificant, implying that they possess a capability for relatively independent evolution by natural selection across different light environments.

Fluctuating asymmetry in Robinia pseudoacacia leaves—possible in situ biomarker?

In this study, we analyzed fluctuating asymmetry (FA) of black locust (Robinia pseudoacacia) leaf traits as a measure of developmental instability in polluted and unpolluted habitats. We aimed to evaluate the potential of this method as a biomarker and its applicability on widely distributed species under in situ conditions. Leaf samples were taken from seven sites—three categorized as unpolluted (natural protected and rural) and four categorized as polluted covering the broad spectrum of intense pollution (industrial and traffic), from 1,489 individual trees in total. Results revealed significant differences in FA with expected higher values in polluted environments. Applicability of FA of R. pseudoaccacia leaf traits as a biomarker for testing potential pollution level, as well as the amount and distribution of sampling effort needed for its application, are discussed.

Phenotypic plasticity and between population differentiation in Iris pumila transplants between native open and anthropogenic shade habitats.

Response to environmental heterogeneity caused by human activity was analyzed on Iris pumila reciprocal transplants between native steppe and anthropogenic (planted pine forest) habitats that were monitored during several growing seasons in a protected area of Deliblato Sand. Morphometric traits exhibited significant plastic responses to the environmental variability between native and anthropogenic habitats that differed in light quantity and quality, as well as in some other ecological indices. Significant differentiation between populations occupying those habitats was also detected. Plastic responses and population differences were substantially related to the variation in general size and had the same direction, plastic responses being larger in magnitude. Estimates of reproductive and vegetative performance of reciprocal transplants detected home site advantage in the native open but not in the secondary shade habitat created under anthropogenic influence.

Ontogeny of flower parts on naturally growing Iris pumila clones: Implications for population differentiation and phenotypic plasticity studies

Previous studies revealed significant phenotypic plasticity, genetic variability and population differentiation of flower morphometric traits on dwarf bearded iris Iris pumila. Also, study of I. pumila flowering phenology revealed significant impact of habitat type as well as population differentiation for flowering time. Since the flowering time can influence other flower traits, we performed this analysis of flower morphometric traits in three time points during the flower bud ontogenic development in two habitat types (open vs. shaded). Analysis revealed that for most of the traits greater trait values were recorded for open habitat but only on latter time points. For most of the analyzed traits direction of differences in bud stage was the opposite to the direction of differences in mature flower stage detected in previous studies. However, length of the stem, a trait that showed the greatest variability between habitats and populations and therefore greatest genetic differentiation and phenotypic plasticity, was significantly greater in the samples from the late flowering shaded habitat in all time samples, indicating that in case of this trait different mechanisms were involved. Those findings have implications for design of the future studies on I. pumila.

Ontogenetic plasticity of anatomical and ecophysiological traits and their correlations in Iris pumila plants grown in contrasting light conditions

To better understand what directs and limits the evolution of phenotype, constraints in the realization of the optimal phenotype need to be addressed. That includes estimations of variability of adaptively important traits as well as their correlation structures, but also evaluation of how they are affected by relevant environmental conditions and development phases. The aims of this study were to analyze phenotypic plasticity, genetic variability and correlation structures of important Iris pumila leaf traits in different light environments and ontogenetic phases, and estimate its evolutionary potential. Stomatal density, specific leaf area, total chlorophyll concentration and chlorophyll a/b ratio were analyzed on I. pumila full‐sib families in the seedling phase and on the same plants after 3 years of growth in contrasting light conditions typical for ontogenetic stage in question. There was a significant phenotypic plasticity in both ontogenetic stages, but significant genetic variability was detected only for chlorophyll concentrations. Correlations of the same trait between different stages were weak due to changes in environmental conditions and difference in ontogenetic reaction norms of different genotypes. Ontogenetic variability of correlation structures was detected, where correlations and integration were higher in seedlings compared with adult plants 3 years later. Correlations were affected by environmental conditions, with integration being higher in the lower light conditions, but correlations between phases being stronger in the higher light treatment. These findings demonstrated that the analyzed traits can be selected and can mostly evolve independently in different environments and ontogenetic stages, with low genetic variability as a potentially main constraint.