Arabidopsis RUP2 represses UVR8-mediated flowering in noninductive photoperiods

Abstract

Plants have evolved complex photoreceptor-controlled mechanisms to sense and respond to seasonal changes in day length. This ability allows plants to optimally time the transition from vegetative growth to flowering. UV-B is an important part intrinsic to sunlight; however, whether and how it affects photoperiodic flowering has remained elusive. Here, we report that, in the presence of UV-B, genetic mutation of REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 (RUP2) renders the facultative long day plant Arabidopsis thaliana a day-neutral plant and that this phenotype is dependent on the UV RESISTANCE LOCUS 8 (UVR8) UV-B photoreceptor. We provide evidence that the floral repression activity of RUP2 involves direct interaction with CONSTANS, repression of this key activator of flowering, and suppression of FLOWERING LOCUS T transcription. RUP2 therefore functions as an essential repressor of UVR8-mediated induction of flowering under noninductive short day conditions and thus provides a crucial mechanism of photoperiodic flowering control.

Keywords: Arabidopsis; UV-B photoreceptor; UVR8; flowering; photoperiodism; plant–environment interaction; sun simulator.

Figure 1.

rup2 flowers early in SDs with UV-B, which is dependent on the UVR8 photoreceptor. (A) Representative images of 100-d-old wild-type (Col), rup1-1, rup2-1, and rup1-1 rup2-1 Arabidopsis plants grown with (+) or without (−) UV-B. (B–E) Quantification of flowering time of wild-type (Col), rup1-1, rup2-1, and rup1-1 rup2-1 plants grown in SDs (B,C) and LDs (D,E) with (+) or without (−) UV-B. (F,G) Quantification of flowering time of wild-type (Col), rup2-1, uvr8-6, and rup2-1 uvr8-6 plants grown in SDs with (+) or without (−) UV-B. The flowering time is represented by total leaf number (rosette and cauline leaves; B,D,F) and days to bolting (C,E,G). Error bars represent standard deviation. n = 30. Shared letters indicate no statistically significant difference in the means. P > 0.05.

Figure 2.

rup2-1 flowers early under realistic irradiation conditions in a sun simulator. Quantification of flowering time of wild-type (Col), rup1-1, rup2-1, uvr8-6, and rup2-1 uvr8-6 plants grown in SDs with (+) or without (−) UV. The flowering time is represented by total leaf number (rosette and cauline leaves; A) and days to bolting (B). Error bars represent standard deviation. n = 20. Shared letters indicate no statistically significant difference in the means. P > 0.05.

Figure 3.

RUP1 and RUP2 interact with CO. (A) Interaction of RUP1 and RUP2 with CO in a yeast two-hybrid growth assay. (Top) Schematic representation of full-length and truncated CO used in interaction analysis. (Bottom) Tenfold serial dilutions of transformed yeast spotted on SD/−Trp/−Leu (DDO; nonselective for interaction) and SD/−Trp/−Leu/−His (TDO; selective) plates. (AD) Activation domain; (BD) binding domain; (EV) empty vector. (B) Colocalization analysis of RUP1-mEGFP and RUP2-mEGFP with either CO-mCherry or NLS-mCherry or without a mCherry fusion protein (−/−) in transiently transformed Nicotiana benthamiana epidermal leaf cells. Shown are confocal images in the GFP and RFP channel as well as the corresponding bright-field and merged images. Bars, 5 µm. (C) Fluorescence lifetime imaging microscopy (FLIM) analyses comparing the different Förster resonance energy transfer (FRET) pairs. (Top) FLIM measurements of transiently transformed N. benthamiana epidermal leaf cells expressing RUP1-mEGFP or RUP2-mEGFP donors in the presence of CO-mCherry or NLS-mCherry acceptor fusion or without a mCherry acceptor (−/−). Error bars indicate standard deviation. n ≥ 20. (***) P ≤ 0.001, a significant difference. (Bottom) Heat maps of representative nuclei used for FLIM measurements. Donor lifetimes of RUP1-mEGFP and RUP2-mEGFP are color-coded according to the scale at the left.

Figure 4.

Early flowering of rup2 in SDs supplemented with UV-B depends on the key flowering regulator CO. (A) Representative images of 100-d-old wild-type (Col), rup2-1, co-101, and rup2-1 co-101 Arabidopsis plants grown with (+) or without (−) UV-B. (B,C) Quantification of flowering time of wild-type (Col), rup2-1, co-101, and rup2-1 co-101 plants grown in SD with (+) or without (−) UV-B. The flowering time is represented by total leaf number (rosette and cauline leaves; B) and days to bolting (C). Error bars represent standard deviation. n = 21. Shared letters indicate no statistically significant difference in the means. P > 0.05.

Figure 5.

Early flowering of rup2 in SDs with UV-B depends on the florigen FT. (A) Quantitative RT–PCR (qRT–PCR) analysis of FT expression in 30-d-old wild-type, rup1-1, rup2-1, rup1-1 rup2-1, and uvr8-6 rup1-1 rup2-1 plants grown under SD + UV on soil. Samples were collected every 3 h; a representative experiment is shown. (ZT) Zeitgeber time; (ZT0) lights on; (ZT8) lights off. (B) GUS assays representing FT promoter activity in 5-d-old wild-type (Col/Pro_FT:GUS), rup2-1/Pro_FT:GUS, uvr8-6/Pro_FT:GUS, and rup2-1 uvr8-6/Pro_FT:GUS seedlings grown in SDs with (+) or without (−) UV-B. (C) Representative images of 100-d-old wild-type (Col), ft-10, rup2-1 ft-10, and rup2-1 Arabidopsis plants grown with (+) or without (−) UV-B. (D,E) Quantification of flowering time of wild-type (Col), ft-10, rup2-1 ft-10, and rup2-1 plants grown in SDs with (+) or without (−) UV-B. The flowering time is represented by total leaf number (rosette and cauline leaves; D) and days to bolting (E). Error bars represent standard deviation. n = 21. Shared letters indicate no statistically significant difference in the means. P > 0.05.

Figure 6.

RUP2 represses CO binding to the FT promoter and inhibits CO-mediated FT expression. (A) qRT–PCR analysis of CO expression in 30-d-old wild-type, rup1-1, rup2-1, rup1-1 rup2-1, and uvr8-6 rup1-1 rup2-1 plants grown under SD + UV on soil. Samples were collected every 3 h; a representative experiment is shown. (ZT) Zeitgeber time; (ZT0) lights on; (ZT8) lights off. (B) GUS assays representing CO promoter activity in 5-d-old wild-type (Col/gCO:GUS) and rup2-1/gCO:GUS seedlings grown in SDs with (+) or without (−) UV-B. (C,D) Quantification of flowering time of wild-type (Col), Col/Pro_35S:3HA-CO, and rup2-1/Pro_35S:3HA-CO plants grown in SDs with (+) or without (−) UV-B. The flowering time is represented by total leaf number (rosette and cauline leaves; C) and days to bolting (D). Error bars represent standard deviation. n = 16. (E) RUP2 does not affect the diurnal regulation of CO stability in Pro_35S:3HA-CO overexpression lines. Immunoblot analysis of 3HA-CO protein level at the indicated Zeitgeber time in 10-d-old Col/Pro_35S:3HA-CO and rup2/Pro_35S: 3HA-CO plants grown in the absence (SD − UV; top panel) or presence (SD + UV; bottom panel) of UV-B. Actin levels are shown as a loading control; wild type (Col) at ZT7 was added as a control sample for anti-HA specificity. (F) HA-CO ChIP-qPCR using 12-d-old wild type (Col), Col/Pro_35S:3HA-CO, and rup2/Pro_35S:3HA-CO seedlings grown in SD + UV (ZT8). The numbers of the analyzed DNA fragments indicate the positions of the 5′ base pair of the amplicon relative to the translation start site. ChIP efficiency of DNA associated with HA-CO is presented as the percentage recovered from the total input DNA (% Input). A representative experiment is shown; error bars represent standard deviation of three technical replicates. (G) Relative LUC activity of protoplasts isolated from co-101 and co-101 rup2-1 plants growing under SD + UV. After protoplast transfection with Pro_FT:fLUC and Pro_35S:CO, chemiluminescence was measured at ZT3–ZT4. Error bars represent standard deviation of four independent experiments, each consisting of at least two independent protoplast transfections.