Proximal disruption of base pairing of the second stem in the upper stem of pri-miR156a caused ambient temperature-sensitive flowering in Arabidopsis

MicroRNAs are generated from primary transcripts (pri-miRNAs) that form hairpin structures. Plant miRNAs play an important role in regulating flowering; however, little is known about the role of their structures in ambient temperature-responsive flowering. We recently showed that disruption of base pairing in the second stem (S2) in the upper stem of pri-miR156a caused hypersensitive flowering in response to ambient temperature changes. To further substantiate our findings on the role of S2 of pri-miR156a, we analyzed the effects of serial disruption (from the proximal or distal sides) of base-pairing in S2 of pri-miR156a on temperature-dependent flowering. We found that flowering time was gradually delayed with increasing size of the proximal disruption of S2 at 16°C. Particularly, disrupting base pairing of 5 nucleotides from the proximal side caused flowering to be hypersensitive to ambient temperature changes, which is similar to the phenotype of plants overexpressing pri-miR156a with a disruption of S2 (156-DBP-S2). However, disrupting base pairing from the distal side did not cause late flowering at 16°C and thus did not cause temperature-sensitive flowering. These results supported our notion that the second stem (S2) in the upper stem of pri-miR156a plays a role in the regulation of ambient temperature-responsive flowering.