ABSTRACT
BACKGROUND: Quantifying gene expression is a critical aspect of applied genomics research across all organisms, and real-time PCR has emerged as a powerful tool for this purpose. However, selecting appropriate internal control genes for data normalization presents specific challenges. This study aimed to identify suitable reference genes for gene expression analysis under various conditions, encompassing salinity, low and high-temperature stresses, and different elicitor treatments. These treatments included titanium dioxide, cold plasma, 24-epibrassinolide, and melatonin, resulting in a total of 13 unique treatments and 148 treatment combinations applied to fenugreek plants. RESULTS: As per the analysis performed with the BestKeeper tool, EEF-1α, and GAPDH were recognized as the most stable reference genes under the majority of conditions. Furthermore, the GeNorm and NormFinder tools identified ß-tubulin and EEF-1α as the most stable reference genes. The findings of this research demonstrated that, although the stability of three reference genes expression was acceptable in almost all evaluated treatments, fluctuations in their expression were observed under the treatments of cold stress with TiO2 NPs application, cold plasma application with salinity stress, and cold plasma application with high-temperature stress compared to others. Simultaneously, the GeNorm analysis results demonstrated that in the mentioned treatments, relying on only one reference gene is inadequate. To corroborate the results, we examined the expression profile of the SSR gene, a pivotal gene in diosgenin biosynthesis, under all investigated treatments and treatment combinations. The outcomes suggested that employing stable reference genes yielded highly consistent results. CONCLUSIONS: The varying expression patterns of the target genes emphasize the crucial need for precise optimization of experimental conditions and selecting stable reference genes to achieve accurate results in gene expression studies utilizing real-time PCR. These findings offer valuable insights into the selection of appropriate reference genes for gene expression analysis under diverse conditions using real-time PCR.
ABSTRACT
Nowadays, it is increasingly crucial to combine innovative approaches with established methods to enhance plant tolerance and maximize the production of beneficial compounds. With this aim in view, a study was carried out to investigate how different melatonin concentrations (0, 30, and 60 ppm), cold plasma treatment (at 3000 and 4000 V), and varying exposure durations (0, 1, 2, and 4 min) affect the physiological and biochemical attributes of fenugreek plants, as well as the levels of diosgenin under salinity stress. This study revealed that the application of 3000 V cold plasma for 2 min with 60 ppm melatonin by establishing cellular redox homeostasis in salinity-treated fenugreek plants, effectively prevented the destruction of pigments and reduced the electrolyte leakage index of malondialdehyde content. The utilization of these two elicitors has the potential to trigger multiple pathways, including the enzymatic and non-enzymatic antioxidants biosynthesis, and abscisic acid-dependent pathways. This activation results in an enhanced production of abscisic acid, auxin, and endogenous melatonin, along with the regulation of signal transduction pathways. Surprisingly, applying these two treatments increased the expression of SQS, CAS, SSR, BGL, SEP, SMT, and diosgenin content by 13, 22.5, 21.6, 19, 15.4, 12, and 6 times respectively. The findings highlight the intricate interplay between these treatments and the positive impact of their combined application, opening up avenues for further research and practical applications in improving plant tolerance to environmental stresses.
