Physiological and Molecular Aspects of Two Thymus Species Differently Sensitive to Drought Stress.

Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative water content (RWC), chlorophyll and carotenoids levels, and mRNA gene expression of metabolic enzymes in Thymus vulgaris (as sensitive to drought) and Thymus kotschyanus (as a drought-tolerant species) were evaluated. The physiological results showed that the treatment predominantly affected the RWC, chlorophyll, and carotenoids content. The gene expression analysis demonstrated that moderate and severe drought stress had greater effects on the expression of histone deacetylase-6 (HDA-6) and acetyl-CoA synthetase in both Thymus species. Pyruvate decarboxylase-1 (PDC-1) was upregulated in Thymus vulgaris at high drought levels. Finally, succinyl CoA ligase was not affected by drought stress in either species. Data confirmed water stress is able to alter the gene expression of specific enzymes. Furthermore, our results suggest that PDC-1 expression is independent from HDA-6 and the increased expression of ACS can be due to the activation of new pathways involved in carbohydrate production.

Biological Response of Lallemantia iberica to Brassinolide Treatment under Different Watering Conditions.

Lallemantia iberica (L. iberica) is an important dry season medicinal plant. Drought, an important abiotic stress, adversely affects the plant's metabolism, which can be alleviated by plant growth regulators like brassinolides. A two-year field experiment was conducted in 2017-2018 to determine the effects of three different irrigation regimes and four brassinolide concentrations on the L. iberica biochemical properties. A split-plot based on a completely randomized block design in three replicates was used as an experimental design with the following irrigation regimes: full watering, watering until flowering and watering until branching. These were the main plots, and 0, 0.5, 1 and 1.5 µM brassinolide concentrations were applied as the subplots. The results showed that many antioxidant enzymes and some biochemical parameters were affected by brassinolide treatment. Furthermore, the highest membrane stability and grain yield were produced in full watering treatment in the second year, and these treatments were not affected by brassinolide application. Several concentrations of brassinolide differently affected the studied treatments, and our study suggests that the amelioration of the effects of the drought stress on L. iberica could possibly be achieved through brassinolide-induced elevation of reactive oxygen species (ROS) scavenging defense systems. There is a need for complementary research to prove the effectiveness of foliar application of this growth regulator to improve the growth and yield of L. iberica under water shortage conditions.

Variation in Terpene Profiles of Thymus vulgaris in Water Deficit Stress Response.

Thyme (Thymus spp.) volatiles predominantly consisting of monoterpenes and sesquiterpenes, serve as antimicrobial, antiseptic and antioxidant in phytomedicine. They also play a key role in plants as secondary metabolites via their potential role against herbivores, attracting pollinators and abiotic stress tolerance. Plant volatiles are affected by different environmental factors including drought. Here, the effect of prolonged water deficit stress on volatile composition was studied on the sensitive and tolerant thyme plant cultivars (T. vulgaris Var. Wagner and T. vulgaris Var. Varico3, respectively). Volatile sampling along with morpho-physiological parameters such as soil moisture, water potential, shoot dry weight, photosynthetic rate and water content measurements were performed on one-month-old plants subsequent to water withholding at 4-day intervals until the plants wilted. The tolerant and sensitive plants had clearly different responses at physiological and volatile levels. The most stress-induced changes on the plants' physiological traits occurred in the photosynthetic rates, where the tolerant plants maintained their photosynthesis similar to the control ones until the 8th day of the drought stress period. While the analysis of the volatile compounds (VOCs) of the sensitive thyme plants displayed the same pattern for almost all of them, in the tolerant plants, the comparison of the pattern of changes in the tolerant plants revealed that the changes could be classified into three separate groups. Our experimental and theoretical studies totally revealed that the most determinant compounds involved in drought stress adaptation included α-phellandrene, O-cymene, γ-terpinene and ß-caryophyelene. Overall, it can be concluded that in the sensitive plants trade-off between growth and defense, the tolerant ones simultaneously activate their stress response mechanism and continue their growth.

FTICR mass spectrometry-based multivariate analysis to explore distinctive metabolites and metabolic pathways: A comprehensive bioanalytical strategy toward time-course metabolic profiling of Thymus vulgaris plants responding to drought stress.

In this research, metabolic profiling/pathways of Thymus vulgaris (thyme) plant were assessed during a water deficit stress using an FTICR mass spectrometry-based metabolomics strategy incorporating multivariate data analysis and bioinformatics techniques. Herein, differences of MS signals in specific time courses after water deficit stress and control cases without any timing period were distinguished significantly by common pattern recognition techniques, i.e., PCA, HCA-Heatmap, and PLS-DA. Subsequently, the results were compared with supervised Kohonen neural network (SKN) ones as a non-linear data visualization and capable mapping tool. The classification models showed excellent performance to predict the level of drought stress. By assessing variances contribution on the PCA-loadings of the MS data, the discriminant variables related to the most critical metabolites were identified and then confirmed by ANOVA. Indeed, FTICR MS-based multivariate analysis strategy could explore distinctive metabolites and metabolic pathways/profiles, grouped into three metabolism categories including amino acids, carbohydrates (i.e., galactose, glucose, fructose, sucrose, and mannose), and other metabolites (rosmarinic acid and citrate), to indicate biological mechanisms in response to drought stress for thyme. It was achieved and approved through the MS signals, genomics databases, and transcriptomics factors to interpret and predict the plant metabolic behavior. Eventually, a comprehensive pathway analysis was used to provide a pathway enrichment analysis and explore topological pathway characteristics dealing with the remarkable metabolites to demonstrate that galactose metabolism is the most significant pathway in the biological system of thyme.

Effect of drought stress on metabolite adjustments in drought tolerant and sensitive thyme.

Drought is one of the most important threats to plants and agriculture; therefore, understanding of the mechanism of drought tolerance is crucial for breeding of drought tolerant plants. Here, we assessed effects of four levels of drought (90%, 55%, 40% and 25% FC) on some physiological criteria and metabolite adjustment of two different drought-responsive thyme plants (Thymus vulgaris as drought sensitive and T. Kotschyanus as drought tolerant species), using 1H-NMR. Among three physiological parameters and 18 identified metabolites, species × treatment effects were significant (P ≤ 0.01) for leaf temperature, acetic acid, citric acid, fumaric acid, malic acid, succinic acid, fructose, sucrose and serine. RWC, chlorophyll and carotenoids content, glucose, alanine and choline were affected by simple effects of species and treatment. Correlation analysis revealed that there is a different correlation between physiological parameters and metabolites in both species. This analysis also revealed that, by ignoring the correlation between malic acid and succinic acid in T. vulgaris, there was no significant correlation between TCA intermediate in both species. According to results, sugars, amino acid and energy metabolism were affected by drought and, among them, TCA intermediates had more alternation in two studied species so, this cycle and its intermediates probably have more prominent role than other identified metabolites in the induction of drought tolerance.

Evaluation and validation of housekeeping genes in two contrast species of thyme plant to drought stress using real-time PCR.

To decrease errors and increase accuracy and reliability of quantitative real-time PCR (qRT-PCR) results, the use of a reference gene is inevitable. Despite the industrial importance of genus Thymus, not any validated reference gene has not been reported for T. kotschyanus and T. vulgaris which could limit such investigations. In this study, the expression stability of seven housekeeping genes including Actin, Cyclophilin-18, elongation factor-1A, glyceraldehyde-3-phosphate dehydrogenase, 18S ribosomal RNA, Cullin, and Polypyrimidine tract-binding protein were evaluated in T. kotschyanus and T. vulgaris which grown at four levels of drought stress using geNorm, NormFinder, and BestKeeper algorithms. Histone deacetylase-6 (HDA-6) gene was also used for validation of evaluated reference genes. In T. vulgaris, all of the algorithms similarly ranked elongation factor-1A and glyceraldehyde-3-phosphate dehydrogenase as the two most stably expressed genes. In T. kotschyanus, only NormFinder and BestKeeper had a similar ranking and identified Actin and glyceraldehyde-3-phosphate dehydrogenase as the two most stably expressed genes, but geNorm algorithm ranked elongation factor-1A and glyceraldehyde-3-phosphate dehydrogenase as the best two reference genes. On the other hand, all algorithms ranked 18S rRNA and Cyclophilin-18 as the least stable genes in T. kotschyanus and T. vulgaris, respectively. Validation results indicated that there was a significant change (0.53-3.19 fold change) in relative expression of HDA-6 normalized by the best stable gene compare to the least ranked gene. Our study presented the first systematic validation of reference gene(s) selection in T. kotschyanus and T. vulgaris and provided useful information to obtain more accurate qRT-PCR results in these species.

Lipidomics Unravels the Role of Leaf Lipids in Thyme Plant Response to Drought Stress.

Thymus is one of the best known genera within the Labiatae (Lamiaceae) family, with more than 200 species and many medicinal and culinary uses. The effects of prolonged drought on lipid profile were investigated in tolerant and sensitive thyme plants (Thymus serpyllum L. and Thymus vulgaris L., respectively). Non-targeted non-polar metabolite profiling was carried out using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry with one-month-old plants exposed to drought stress, and their morpho-physiological parameters were also evaluated. Tolerant and sensitive plants exhibited clearly different responses at a physiological level. In addition, different trends for a number of non-polar metabolites were observed when comparing stressed and control samples, for both sensitive and tolerant plants. Sensitive plants showed the highest decrease (55%) in main lipid components such as galactolipids and phospholipids. In tolerant plants, the level of lipids involved in signaling increased, while intensities of those induced by stress (e.g., oxylipins) dramatically decreased (50-60%), in particular with respect to metabolites with m/z values of 519.3331, 521.3488, and 581.3709. Partial least square discriminant analysis separated all the samples into four groups: tolerant watered, tolerant stressed, sensitive watered and sensitive stressed. The combination of lipid profiling and physiological parameters represented a promising tool for investigating the mechanisms of plant response to drought stress at non-polar metabolome level.

Comprehensive list of metabolites measured by DI-FTICR mass spectrometry in thyme plants with contrasting tolerance to drought.

This article contains data related to the main research entitled "Metabolomic approach reveals the biochemical mechanisms underlying drought stress tolerance in Thyme" (Moradi et al., 2017) [1]. Two thyme populations with contrasting drought tolerance were subjected to long term water deficit. Leaf samples harvested at the end of stress period and bi-phasic extraction carried out to get polar and non-polar fractions. Extracted samples were analyzed through Direct Infusion FT-ICR mass spectrometry. Date files comprise of four separate tables for all the putatively identified metabolites and their intensities in watered and droughted plants. P-values beside each m/z values indicate significances of difference between peak intensities of stressed and control conditions.

Metabolomic approach reveals the biochemical mechanisms underlying drought stress tolerance in thyme.

Thyme as a perennial herb has been recognized globally for its antimicrobial, antiseptic and spasmolytic effects. In this investigation, we have used non-targeted metabolite and volatile profiling combined with the morpho-physiological parameters in order to understand the responses at the metabolite and physiological level in drought sensitive and tolerant thyme plant populations. The results at the metabolic level identified the significantly affected metabolites. Significant metabolites belonging to different chemical classes consisting amino acids, carbohydrates, organic acids and lipids have been compared in tolerant and sensitive plants. These compounds may take a role through mechanisms including osmotic adjustment, ROS scavenging, cellular components protection and membrane lipid changes, hormone inductions in which the key metabolites were proline, betain, mannitol, sorbitol, ascorbate, jasmonate, unsaturated fatty acids and tocopherol. Regarding with volatile profiling, sensitive plants showed an increased-then-decreased trend at major terpenes apart from alpha-cubebene and germacrene-D. In contrast, tolerant populations had unchanged terpenes during the water stress period with an elevation at last day. These results suggesting that the two populations are employing different strategies. The combination of metabolite profiling and physiological parameters assisted to understand precisely the mechanisms of plant response at volatile metabolome level.

Buxtonella spp. like infection in cattle in Sanandaj province, Iran.

Buxtonellosis is a disease caused by the ciliated protozoan Buxtonella sulcata (Jameson, 1926). B. sulcata is a common protozoan of ruminants and may result in subclinical infection or clinical disease including diarrhea. This study examined the prevalence of B. sulcata in cattle from Sanadaj province, Iran. Faecal samples were collected from cattle in the province from May 2013 to June 2014. A total of 217 cattle were selected randomly according to the age, sex, health, management system and season. In total, 99 cattle (45.63%) were found to be infected with B. sulcata. In adults, prevalence of B. sulcata infection (51.64%) was higher than calves (28.58%) and young cattle (40.55%). The prevalence of B. sulcata infection was also found to be higher in female (47.32%) than male (38.46) cattle. B. sulcata infection was significantly (p<0.05) higher in poor health cattle (body condition and weight) (79.54%) than healthy cattle (24.47%). This study demonstrates that cattle are highly susceptible to B. sulcata infection under a variety of housing and environmental conditions in this region of Iran. The study also demonstrates a significant effect of infection on cattle health.

Investigation of DNA-cationic bolaform surfactants interaction with different spacer length.

In this paper interaction of DNA with cationic bolaform surfactants is investigated. The structural formula for synthesized bolaforms is as follows: bolaform B1 with structural formula Br(-)(CH3)3N(+)(CH2)3N(+)(CH3)Br(-) and bolaform B2 with structural formula of Br(-)(CH3)3N(+)(CH2)12N(+)(CH3)Br(-). There are stronger electrostatic interactions in bolaform B1 due to shorter spacer length, while there are stronger hydrophobic interactions in bolaform B2 compared to bolaform B1 due to existence of 12 carbons in hydrocarbonic chain. The structure of bolaforms consists of two polar head groups which play important role in DNA compaction. Surface tension change in aqueous solution of bolaform surfactants is measured using tensiometer. Electrical conductivity of surfactants aqueous solution is examined with and without DNA. DNA compaction is tracked in the presence of bolaforms by dynamic light scattering (DLS) technique. Results of DLS indicate bolaforms with shorter spacer length (dominant electrostatic interactions) are more influential in compressing DNA compared to bolaforms with longer spacer length (stronger hydrophobic interactions). UV-vis and fluorescence spectroscopies specify the binding mechanism of bolaform surfactants to DNA.