Effect of ultrasonic waves on crocin and safranal content and expression of their controlling genes in suspension culture of saffron (<i>Crocus sativus </i>L.)

<p>The expression of biosynthesis controlling genes of crocin and safranal in saffron (<i>Crocus sativus</i>) can be influenced by ultrasonic waves. Sterilized saffron corms were cultured in a ½-MS medium supplemented by 2-4-D and BAP.  Saffron callus cells were treated with ultrasonic waves in a cellular suspension culture under optimal growth conditions. The samples were collected at 24 and 72 hours after treatment in three replications. The secondary metabolites were measured by high-performance liquid chromatography and the gene expression was analysed by the real-time polymerase chain reaction. Results indicate that this elicitor can influence the expressions of genes <i>CsBCH, CsLYC</i> and <i>CsGT</i>-<i>2</i>; the ultrasonic waves acted as an effective mechanical stimulus to the suspension cultures. The analysis of variance of the ultrasonically produced amounts of safranal and crocin indicates that there is a significant difference between once- and twice-treated samples in that the amount of safranal was the highest within the samples taken from the twice-treated suspension culture at 72 h after the ultrasound treatment, and the crocin was maximised after 24 h passed the twice-applied ultrasound treatment.</p

List of eugenol-sensitive gene deletion mutants from GDA analysis that showed greater than 70% reduction in colony size.

<p>List of eugenol-sensitive gene deletion mutants from GDA analysis that showed greater than 70% reduction in colony size.</p

Auxotroph supplement assay shows that eugenol inhibits the functions of aromatic and branched-chain amino acid transporters.

<p>Strains of <i>S. cerevisiae</i> from GDA library were selected with gene deletions in the tryptophan, phenylalanine, tyrosine (A) or isoleucine (B) biosynthesis pathways. Cultures were 10-fold serially diluted and spotted on synthetic medium supplemented with tryptophan, phenylalanine, tyrosine and isoleucine, either containing a subinhibitory concentration of eugenol (0.18 mg/ml) or without eugenol (control). The plates were incubated at 30°C for 1–2 days and then photographed.</p

Nearly half of eugenol-sensitive strains have deletions in genes involved in aromatic and branched-chain amino acid synthesis or uptake.

<p>(A) The haploid non-essential yeast gene deletion array was subjected to a subinhibitory concentration of eugenol. Colony size reduction was used to detect sensitivity. The mutants most sensitive to eugenol were clustered according to the cellular processes in which their deleted genes participated. (B) Eugenol-sensitive strains identified by GDA were verified by drop out plates. Wild type and eight randomly selected gene deletion mutant strains that were eugenol-sensitive based on GDA analysis were 10-fold serially diluted and spotted on solid YPD medium with a subinhibitory concentration (0.18 mg/ml) of eugenol or without eugenol (control). The plates were incubated at 30°C for 1–2 days and then photographed. All deletion mutants selected exhibited increased sensitivity to eugenol, providing verification of the GDA analysis.</p

Phenotypic suppression assay shows that Tat1p and Gap1p permeases are targets of eugenol in the yeast cytoplasmic membrane.

<p>Overexpression constructs of four permeases, <i>BAP2, GAP1</i>, <i>TAT1</i> and <i>TAT2</i> were transformed separately into the <i>aro1Δ</i> strain. Transformants were grown overnight in SC-ura medium, diluted 1∶300 and then added to each well of a sterile microtiter plate with or without 0.18 mg/ml eugenol. Growth of the <i>aro1Δ</i> transformants containing the overexpression constructs were compared to one with a control plasmid by measuring the optical density of cells in each well (OD₆₀₀). The values are expressed as mean (±SD, n = 3) and significant differences between treatment and plasmid control are indicated as <i>P<0.05</i> (*) and <i>P<0.01</i> (**) based on Student T-test. Inset: eugenol is structurally similar to aromatic amino acids and is synthesized in plants via the phenylpropanoid pathway from phenylalanine.</p

Eugenol does not induce leakage of liposomes made of total yeast lipids or inhibit protein synthesis.

<p>(A) Release of carboxyfluorescein from large unilamellar vesicles (LUVs) over a series of eugenol concentrations (0.003 to 1.6 mg/ml, bottom axis) was compared to 100% release from liposome exposed to Triton-X 100. <i>Echinacea purpurea</i> extract was used as a positive lysis control over a concentration gradient of 0.01 to 5% (top axis). Data correspond to the mean % leakage values (±SD) of three independent experiments. (B) Yeast exposed to subinhibitory (0.21 mg/ml) or inhibitory (0.27 mg/ml) concentrations of eugenol do not have significantly decreased β-galactosidase activity in comparison to the untreated control. In contrast, the inhibitor of protein translation, cycloheximide, significantly reduced β-galactosidase activity in the assay. These observations indicate that eugenol does not reduce efficiency of translation in yeast as would be expected for compounds that perturb the intracellular pool of amino acids. The values are expressed as mean ±SD of triplicates, difference between treatment and untreated control are indicated as p<0.05 (*) and p<0.01 (**).</p