Verification of the function of tae-miR399 in the freezing tolerance in transgenic Arabidopsis thaliana
To describe the function of tae-miR399 in tolerance against freezing stress, we constructed it into an expression vector under the control of the cauliflower mosaic virus 35S promoter (OEmiR399). After producing Arabidopsis transgenic plants through several cycles of kanamycin resistance selection, PCR analysis of genomic DNA with the primers specific to tae-miR399 and 35S corroborated 9 independent transgenic lines (Fig. S3A). The total RNA of these 9 Arabidopsis lines was extracted for their miR399 expression level by PCR semi-quantitative detection, and the wild type Arabidopsis plant (WT) was used as a control. The results showed that miR399 was expressed in all OEmiR399 transgenic Arabidopsis plants. Although WT also has miR399 endogenously, the expression of miR399 in OEmiR399 transgenic Arabidopsis plants was significantly higher than that in WT. Two lines (OEmiR399-4 and OEmiR399-6) were selected and self-crossed to obtain stable homozygous lines.
To investigate the effects of tae-miR399 on tolerance to freezing stress in Arabidopsis, one-month-old Arabidopsis plants (WT, OEmiR399-4 and OEmiR399-6) were transferred to the 4°C for 3 d for cold acclimation and then the temperature dropped to -10°C for 2 h. Before treatment, there was no obvious difference in the morphological parameters including synchronous growth, leaf size and color among these Arabidopsis lines (Fig. 4A), and the primary roots of OEmiR399 lines were significantly longer than WT (Fig. 4B).After the cold treatment, the leaves of all Arabidopsis lines curled as the temperature decreased. Obviously, the leaf color of WT is darker than that of OEmiR399 lines. Moreover, the tae-miR399 overexpressing plants showed higher survival rates than the rest after recovering culture (Fig. 4C). To verify whether tae-miR399 improved the freezing tolerance of Arabidopsis, physiological indicators of all Arabidopsis lines were determined. The results showed that there was no significant difference in electrical conductivity and MDA content among all lines at 24°C (Fig. 4D and E). With the decrease of temperature, the electrical conductivity and MDA content of all lines increased. At 4°C and -10°C, the MDA content of OEmiR399 lines were lower than that of WT. The electrical conductivity of OEmiR399 lines were lower than that of WT at -10°C (Fig. 4D and E). Generally, the OEmiR399 lines showed better physiology performance under freezing treatment as compared to other lines.
qRT-PCR analysis of miR399 and AtUBC24 in all Arabidopsis lines showed that the expression of miR399 in OEmiR399 transgenic Arabidopsis plants was always significantly higher than that of WT, and the expression of its target gene AtUBC24 was always significantly lower than that of WT (Fig. 5A and B). Moreover, at -10°C, the expression of miR399 in WT significantly increased and the expression ofAtUBC24 in WT decreased obviously. At 4°C, the expression of miR399 in OEmiR399 transgenic Arabidopsis plants decreased and the expression of AtUBC24 in OEmiR399 transgenic Arabidopsis plants increased (Fig. 5A and B).