Arabidopsis Cold Stress Tolerance Improvement via AthHOS1-targeting HOS1-amiRNA Approach

In this study, we examined the efficacy of the artificial microRNAs (amiRNAs) technology in targeting the HOS1 gene for the enhancement of cold stress tolerance in Arabidopsis thaliana Ler-0 ecotype. The impact of athHOS1-amiRNA overexpression on the response of transgenic plants to cold stress was assessed using RT-qPCR in 3-week-old seedlings of the T3 generation. Additionally, the response of wild-type plants of the same age to cold stress (4oC) for various durations (6, 12, 24, 48, and 96 hours) was also evaluated. Comparative analysis revealed that athHOS1-amiRNA downregulated athHOS1 in transgenic plants after prolonged exposure to low temperature (48 h and 96 h) (Pearson’s correlation coefficient of -0.407; P< 0.05). Interestingly, while prolonged cold stress at 96 h led to a significant upregulation of athHOS1 in wild-type plants, the suppression of athHOS1-amiRNA in transgenic plants disrupted the expected circadian rhythm of athHOS1 by preventing its upregulation. Furthermore, T3 plants that had been cold-acclimated exhibited a 17% increase in freezing tolerance (-1 to -8°C) compared to wild-type plants, indicating the success of this approach in enhancing Arabidopsis tolerance to low temperatures, at least in the Ler-0 ecotype. In order to gain a deeper understanding of the intricate dynamics of gene/protein networking during cold acclimation and its interaction with the athHOS1-amiRNA approach, further characterization is required. This includes measuring the expression levels and half-life of athHOS1-amiRNA and HOS1 mRNA, as well as evaluating the protein level of HOS1 and its direct targets, such as ICE1, in different Arabidopsis ecotypes and at different time intervals of low temperature exposure.