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

ABSTRACT

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.

Abbreviations

S2

second stem

EV

empty vector

LNR

Leaf number ratio

DBP

disruption of base-pairing

MicroRNAs (miRNAs) are non-coding RNAs of ∼21 nucleotides. They negatively control expression of their target genes via sequence-specific degradation or translational repression.¹ MiRNAs are generated from precursor primary miRNAs (pri-miRNA) via double cleavage events catalyzed by DICER-LIKE 1 (DCL1), which forms a complex with HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE).² Mature miRNAs are then protected by HUA ENHANCER1 (HEN1)³ and exported to the cytoplasm by HASTY (HST)⁴. Subsequently, one strand of the miRNA/miRNA* duplex guides the ARGONAUTE complex to its target RNAs.⁵

The secondary structure of the pri-miRNA plays an important role in processing of plant miRNAs including miR172a, miR171a, miR390a, and miR319a.^6-10 Previously, we identified structural determinants that are important for the processing of pri-miR156a in temperature-responsive flowering.¹¹ We have shown that structural variations in the upper stem, which is adjacent to the first cleavage site of pri-miR156a, affected pri-miR156a processing and ambient temperature-responsive flowering in Arabidopsis. The second stem (S2) adjacent to the first cleavage of pri-miR156a plays an especially important role. Disruption of base pairing of S2 in pri-miR156a (156-DBP-S2), in which opening S2 made a large bulge along with adjacent bulges, caused weak late flowering at a normal temperature (23°C), but caused strong late flowering at a low temperature (16°C), resulting in flowering that was hypersensitive to changes in ambient temperature.¹¹

To further dissect the structural determinants of S2 in the upper stem of pri-miR156a, we generated transgenic plants overexpressing mutations that serially disrupted base pairing of S2. The name of each variant contains the miR156-DBP-S2, which indicates disruption of base pairing (DBP) in S2, followed by the direction of the serial mutation (P: proximal; D: distal) and the number of bases disrupted (Fig. 1A and 1B). For instance, 156-DBP-S2P2 indicates a mutation that disrupts the base pairing of the proximal 2 nucleotides in the S2 of pri-miR156a; this enlarges the bulge B2 and reduces S2. Likewise, 156-DBP-S2D3 indicates a mutation that disrupts the base pairing of the distal 3 nucleotides in S2 of pri-miR156a, enlarging B1 and reducing S2.

Figure 1.

Analysis of structural variants in stem 2 (S2) in pri-miR156a. (A) Secondary structure of the pri-miR156a hairpin. The mature miR156a and miR156a* sequences are highlighted in cyan and purple, respectively. B, L, and S denote bulge, loop, and stem sequences, respectively. (B) A diagram of structural variants with serial disruptions of base pairing of S2 of pri-miR156a. Partial miR156a/miR156a* sequences are shown. Mutated nucleotides for disrupting base pairing are shown in red. (C and D) Total leaf numbers of homozygous plants carrying each disruption of base pairing in S2 of pri-miR156a at 23°C (C) and 16°C (D). The flowering time of 156-DBP-S2 plants is adapted from our previous study.¹¹ Distribution of leaf numbers is shown as a box plot.¹³ The center lines show the medians; box limits indicate the 25^th and 75^th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range (IQR) from the 25^th and 75^th percentiles, outliers that exceeded the 1.5x IQR are represented by dots. EV: empty vector control (E) Leaf number ratio (16°C/23°C) (LNR) of plants overexpressing structural variants of pri-miR156a. The LNR of 156-DBP-S2 plants is adapted from our previous study¹¹. Distribution of LNR of transgenic plants used in this study is presented as a box plot at the bottom of graph. The solid lines indicate the trend line. EV: empty vector control; O/X: plants overexpressing the un-mutated construct (35S::miR156a).

To study the in vivo effect of these mutations, we analyzed flowering time changes caused by overexpression of each mutation, measuring total leaf numbers produced when the height of the primary inflorescence of transgenic plants reached 5 cm. At 23°C, regardless of the mutation type, all mutants flowered earlier than the control 35S::miR156a plants (19.6 leaves). They showed intermediate flowering times between the plants expressing empty vector (EV) (11.0 leaves) and pri-miR156a (19.6 leaves) (Fig. 1C).¹¹ At 16°C, all transgenic plants also flowered earlier than 35S::miR156a plants (50.8 leaves), as seen at 23°C. An interesting observation was that flowering time was gradually delayed with increasing size of the proximal disruption of S2 at 16°C. Total leaf numbers of 156-DBP-S2P1, 156-DBP-S2P2, 156-DBP-S2P3, and 156-DBP-S2P4 plants were 16.4, 23.3, 23.7, and 23.7, respectively. Moreover, the 156-DBP-S2P5 plants, which have disrupted base pairing of the proximal 5 nucleotides of S2, exhibited strongly delayed flowering (33.3 leaves). This flowering time was closest to that seen in transgenic plants carrying 156-DBP-S2 (46.9 leaves), in which opening of S2 created a large bulge along with B1 and B2 (Figs. 1D and 2).¹¹ In the case of distal mutations, such a gradual increase in total leaf numbers was very weak, although 156-DBP-S2D5 plants flowered with 23.1 leaves (c.f. 156-DBP-S2D2 plants: 16.1 leaves). These results suggested that the effect on flowering time was more pronounced for the proximal disruption than for the distal disruption of S2 in pri-miR156a (Fig. 1D and 2).

Figure 2.

Flowering phenotypes of mutants carrying serial disruptions of S2 of pri-miR156a at 16°C. Note that a larger proximal disruption in S2 caused delayed flowering at 16°C, resulting in increased sensitivity to ambient temperature changes. The mature miR156a and miR156a* sequences are highlighted in cyan and purple, respectively. B, L, and S denote bulge, loop, and stem, respectively.

Next, to determine whether the serial disruption of S2 caused altered responses to changes in ambient temperature, the temperature sensitivity was analyzed by calculating the leaf number ratio (LNR: the number of leaves at flowering at 16°C, divided by the number of leaves at 23°C).¹² The 35S::miR156a plants showed LNR values of 2.6 (c.f. EV plants: 2.0), indicating that miR156a overexpression caused flowering to be hypersensitive to ambient temperature changes (Fig. 1E). Previously, we showed that the 156-DBP-S2 mutation caused dramatic hypersensitivity to ambient temperature, producing a LNR value of 3.6 (Fig. 1E).¹¹ The average LNR (1.8) of transgenic plants carrying serial disruptions of pri-miR156a S2 was lower than that of 35S::miR156a plants, indicating that disruptions of S2 in the pri-miR156a decreased sensitivity to ambient temperature changes. Interestingly, 156-DBP-S2P5 plants showed a LNR value of 2.5, which was similar to that seen in plants overexpressing pri-miR156a (LNR: 2.6), which showed ambient temperature-sensitive flowering, and close to that seen in 156-DBP-S2 plants (LNR: 3.6). Our results suggested that proximal disruption of base pairing in S2 exerted an effect closer to the effect of disruption of all of S2 (156-DBP-S2) (Fig. 1E).

In this study, we investigated the effect of serial disruptions in stem 2 adjacent to the first cleavage site of pri-miR156a on the control of ambient temperature-responsive flowering. Our data presented here suggest that the proximal side of S2 is important for the role of the S2 in ambient temperature-responsive flowering (Fig. 2).

Disclosure of potential conflicts of interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by the Creative Research Initiative Program (2008-0061988 to J.H.A) through the National Research Foundation of Korea funded by the Korean Government (MSIP) and a Korea University Grant.