Variation of growth indices and expression of 1- deoxyxylulose 5-phosphate reductase isomerase (DXR) gene under the effect of salicylic acid and chitosan in Thymbra spicata L. with different irrigation levels

Thymbra spicata L. belongs to the Lamiaceae family and is a rich source of aromatic compounds and essential oils. This research was conducted to study the effect of salicylic acid and chitosan elicitors on the growth indicators and DXR gene expression in T. spicata L. under different levels of irrigation towards enhancing the performance of this medicinal plant in experimental pots in a greenhouse in Ilam province during 2017-18. A factorial design was used with 15 treatments and three replications in a completely randomized study. Irrigation regimes at three levels of normal (A₁), 70% of field capacity (A₂), 40% of field capacity (A₃), and foliar spraying at five levels, including control (B₁), chitosan at concentrations of 0.5 g.l-1 (B₂) and 1 g.l-1 (B₃), salicylic acid foliar spraying at concentrations of 2.5 mM (B₄) and 5.0 mM (B₅) were used at the 10-12 leaf stage. To prepare chitosan and salicylic acid elicitors, the powders were dissolved in 5% acetic acid and then adjusted to desired concentrations with ddH2O. Irrigation regimes were applied three months after cultivation (15-20 cm seedlings). To apply the elicitors simultaneously with the water stress treatment, foliar spraying was done three times at ten-day intervals. Quantitative RT-PCR was used to investigate the expression changes of the DXR gene, which is one of the key genes in the carvacrol biosynthesis pathway. In this regard, total RNA was extracted, and cDNA was synthesized after assessment of its quantity and quality. With the aid of specific primers, the target sequence was amplified. Furthermore, using the GAPDH reference gene reported in Hyssop thyme, the gene expression level was investigated through the relative quantification method. The method of difference in cycle threshold (2-∆∆CT) and Relative Expression Software Tool (REST) were used to analyze the data. The analysis of the variance table showed the effect of different levels of irrigation on all quantitative growth indices, including plant height, root length, number of main branches, root fresh and dry weight, leaf fresh weight, stem fresh and dry weight, except leaf dry weight, were significant (p<0.01). The interactive effect of low irrigation and foliar spray of salicylic acid and chitosan on root fresh weight, leaf fresh weight, and stem fresh weight was significant at 1%. Accordingly, the interaction effect of irrigation and foliar spraying on the dry weight of the stem was significant at the 5% probability level. The results showed that the interactive effect of irrigation and foliar spray on leaf dry weight, number of main branches, and root length was insignificant. In this study, DXR gene expression did not show many changes after applying two different levels of chitosan (0.5 and 1.0 g.l-1). In contrast, salicylic acid treatment with 2.5 mM was significant at 1% probability and caused an increase in gene expression. The transcript level of this gene increased 29.72 times after applying 5.0 mM of this solution, which was significant at the 1% level. The amount of low irrigation decreased this gene's expression. Based on the obtained data, the normal irrigation method is the most suitable for hyssop thyme plants regarding their growth indicators. Foliar spraying of chitosan at different levels did not significantly affect growth indices and DXR gene expression. At the same time, salicylic acid increased them in this species. As a result, salicylic acid elicitor can increase carvacrol yield and biosynthesis.