Abstract
In this study the artificial microRNAs (amiRNAs) technology targeting HOS1 gene was tested for its applicability for the improvement of cold stress tolerance in Landsberg-0 (Ler-0) ecotype of Arabidopsis thaliana. The chosen approach was designed to suppress AtHOS1 gene expression through the overexpression of amiRNA-HOS1. The effect of AtHOS1-amiRNA overexpression to transgenic plants’ response to cold stress was determined by Real Time PCR. The expression levels of amiRNA and its target, AtHOS1 gene, were observed in 3-week old seedlings of T3 generation and in wild-type plants after 6h, 12h, 24h, 48h and 96h of their exposure to cold stress (4ºC). Comparative analysis revealed that AtHOS1-amiRNA negatively regulated AtHOS1 in transgenic plants upon plants lengthen exposure (for 48h and 96h) to low temperature (Pearson’s correlation coefficient of -0.407; P < 0.05). Even though prolonged cold stress caused extended up regulation of AtHOS1 in wild type plants, in transgenic plants AtHOS1-amiRNA suppression disturbed expected AtHOS1 circadian rhythm by preventing further AtHOS1 up regulation. Moreover, transgenic plants showed AtHOS1 down regulation 96h after the cold stress onset, due to sufficient overexpression of AtHOS1-amiRNA, which allowed cold signaling amplification in transgenic plants. As a result of that, cold-acclimated transformed plants displayed 17% higher freezing tolerance (-1°C to -8°C) in comparison to wild type plants, demonstrating the success of chosen approach in improving Arabidopsis tolerance to low temperatures, at least in Ler-0 ecotype.
Alteration of [beta]-Tubulin Gene Expression during Low-Temperature Exposure in Leaves of Arabidopsis thaliana