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.

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.

Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR.

BACKGROUND: Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a "gold standard" for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. METHODS: Five milliliter blood samples from healthy volunteers were spiked with 10(0)-10(6) C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. RESULTS: No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10(0) CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C. CONCLUSION: The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.