Internal and environmental cues, including ambient temperature changes, regulate the timing of flowering in plants. Arabidopsis miR156 represses flowering and plays an important role in the regulation of temperature-responsive flowering. However, the molecular basis of miR156 processing at lower temperatures remains largely unknown. Here, we performed nuclear magnetic resonance studies to investigate the base-pair opening dynamics of model RNAs at 16 degrees C and investigated the in vivo effects of the mutant RNAs on temperature-responsive flowering. The A9C and A10CG mutations in the B5 bulge of the lower stem of pri-miR156a stabilized the C15.G98 and U16.A97 base-pairs at the cleavage site of primiR156a at 16 degrees C. Consistent with this, production of mature miR156 was severely affected in plants overexpressing the A9C and A10CG constructs and these plants exhibited almost no delay in flowering at 16 degrees C. The A10G and A9AC mutations did not strongly affect C15.G98 and U16.A97 base-pairs at 16 degrees C, and plants overexpressing A10G and A9AC mutants of miR156 produced more mature miR156 than plants overexpressing the A9C and A10CG mutants and showed a strong delay in flowering at 16 degrees C. Interestingly, the A9AC mutation had distinct effects on the opening dynamics of the C15.G98 and U16.A97 base-pairs between 16 degrees C and 23 degrees C, and plants expressing the A9AC mutant miR156 showed only a moderate delay in flowering at 16 degrees C. Based on these results, we propose that fine-tuning of the base-pair stability at the cleavage site is essential for efficient processing of pri-miR156a at a low temperature and for reduced flowering sensitivity to ambient temperature changes. (C) 2017 Elsevier Inc. All rights reserved.