Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica.
As sessile organisms, plants are continuously exposed to a wide range of environmental stress. In addition to their crucial roles in plant growth and development, small signaling peptides are also implicated in sensing environmental stimuli. Notably, recent studies in plants have revealed that small signaling peptides are actively involved in controlling stomatal aperture to defend against biotic and abiotic stress. This review illustrates our growing knowledge of small signaling peptides in the modulation of stomatal aperture and highlights future challenges to decipher peptide signaling pathways in guard cells.
An effective method for the ultrasound-assisted extraction of indigo and indirubin from Fort. was established and their antioxidant activities were investigated. Response surface methodology based on a three-level, four-factor Box-Behnken design was used to optimize the extraction conditions. Analysis of variance showed that the quadratic model was significant for the extraction of indigo and indirubin (112.72% ± 1.65% and 116.42% ± 1.27%, respectively) under the optimal conditions (methanol concentration, 80%; extraction time, 25 min; ratio of solid to liquid, 1:34 g/mL; and extraction temperature, 41 °C) and was in good agreement with the predicted value. Moreover, evaluation of the antioxidant activities suggested that indigo and indirubin presented better scavenging effects on 1,1-diphenyl-2-picrylhydrazyl free radical and superoxide radical than the extract and the extract revealed certain antioxidant activities in hydroxyl radical scavenging and reducing power, and indigo and indirubin could be used as natural antioxidants in the food or medicine industry.
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