Phytochrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice

Abstract

Phytochromes confer the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. To better understand the molecular mechanisms of day-length recognition, we examined the interaction between phytochrome signals and circadian clocks in photoperiodic-flowering mutants of rice. Monitoring behaviors of circadian clocks revealed that phase setting of circadian clocks is not affected either under short-day (SD) or under long-day (LD) conditions in a phytochrome-deficient mutant that shows an early-flowering phenotype with no photoperiodic response. Non-24-hr-light/dark-cycle experiments revealed that a rice counterpart gene of Arabidopsis CONSTANS (CO), named PHOTOPERIOD SENSITIVITY 1 (Heading date 1) [SE1 (Hd1)], functions as an output of circadian clocks. In addition, the phytochrome deficiency does not affect the diurnal mRNA expression of SE1 upon floral transition. Downstream floral switch genes were further identified with rice orthologs of Arabidopsis FLOWERING LOCUS T (FT). Our RT-PCR data indicate that phytochrome signals repress mRNA expression of FT orthologs, whereas SE1 can function to promote and suppress mRNA expression of the FT orthologs under SD and LD, respectively. This SE1 transcriptional activity may be posttranscriptionally regulated and may depend on the coincidence with Pfr phytochromes. We propose a model to explain how a short-day plant recognizes the day length in photoperiodic flowering.

Figure 1

Circadian clock behavior in the se5 mutant. (DD) 24 h darkness; (LL) 24 h light; (WT) wild type. (A) Rhythmic mRNA expression of two rice ccgs, OsLHY and CAB1R, under constant light conditions in RNase protection assays. RNA samples were harvested every 2 h from leaves of 1-month-old rice plants. Leaves from three to five plants were mixed. (Left, DD) After 5 d of entrainment (12L12D). (Right, LL) After the same entrainment. A rice actin or ubiquitin fragment was used as a control probe. (B) Diurnal mRNA expression of OsLHY under LD and SD conditions in RNase protection assays. RNA samples were harvested every 2 h. (Top) RNase protection assay. The ubiquitin is a control probe. (Bottom) Normalized data. (C) Rhythmic cab1r::luc expression in DD after SD and LD entrainments. Rhythmic cab1r::luc expression in 7-day-old rice seedlings was measured after 5 d of entrainment (10L14D for SD entrainment; 14L10D for LD entrainment). (Left, DD) After SD entrainment. (Right, LL) After LD entrainment. Averages of six or seven measurements were plotted with standard deviations. Representative of two or three independent experiments.

Figure 2

Circadian clock behavior and flowering responses in non-24-h photoperiods. (A) Rhythmic cab1r::luc expression in 24L12D and subsequent DD. Five-day-old seedlings were used for measurements. Averages of six seedlings are plotted with standard deviations. Representative of two experiments. (B) Rhythmic cab1r::luc expression in 36L12D. Five-day-old seedlings were used. Averages of six seedlings are plotted with standard deviations. (C) Rhythmic cab1r::luc expression in 24L. Five-day-old seedlings were used. Averages of six seedlings are plotted with standard deviations. Plants were grown under the 24-h temperature cycle (12 h at 25°C/12 h at 30°C) shown at the top. (D) Flowering time in non-24-h photoperiods. Heading dates after sowing were measured as flowering times. Averages were plotted with standard deviations. Plant numbers are shown in parentheses. Representative of two or three experiments. (se5) se5 mutant with Norin8 genetic background; (se1) se1 mutant with Ginbouzu genetic background; (N8) cv. Norin 8; (GIN) cv. Ginbouzu. Lines 1, 2, and 3 represent se1 se5 mutant lines derived from three F₂ se1 se5 plants.

Figure 3

SE1 mRNA expression in se5 mutant. (A) Diurnal SE1 mRNA expression under long-day (LD) conditions by RT-PCR analysis. RNA samples were harvested from 20-day-old rice plants every 2 h from leaves of plants grown under LD conditions after sowing. Leaves from three to five plants were mixed into one sample. The DNA blot was hybridized with specific radioactive probes to perform a quantitative analysis. Radioactive signals were quantified with a BAS 2000 image analyzer. The normalized data for ubiquitin expression are plotted. Average values and standard deviations from three RT-PCR data are shown. Representative of two independent experiments. (B) Diurnal SE1 mRNA expression under short-day (SD) conditions by RT-PCR analysis. RNA samples were harvested from 28-day-old rice plants in the same way as in A. One RNA sample of se5 in SD conditions is missing, so we do not show one data at 18:00. The DNA blot was hybridized with specific radioactive probes to perform a quantitative analysis. Radioactive signals were quantified with a BAS 2000 image analyzer. The normalized data for ubiquitin expression are plotted. Average values and standard deviations from four RT-PCR data are shown. Representative of two independent experiments.

Figure 4

FT-like gene family in rice and phenotypes of UBQ::FTL plants. (A) Amino acid comparison among rice FT-like genes. Two genes, FT and TSF, belong to the FT family in Arabidopsis. Only amino acids that are not conserved are highlighted. (B) A phylogenic analysis of the FT-TFL family in Arabidopsis and rice. All six members in Arabidopsis are shown with (At) in this tree. Clustal W and NJ methods were used for the alignment and phylogenic tree, respectively. Bootstrap values are shown at branches. (C) A regenerated rice plant transformed with UBQ::FTL. A terminal flower is visible at the tip of the plant (arrowhead). All axial buds produced several leaves with terminal tissue. Bar, 1 cm. (D) Magnification of the terminal flower in C. There is a pistil-like structure at the center (arrowhead). A root was developed at the base of this floret (arrow). Bar, 1 mm. (E) Cross-section of the terminal flower in C. Stamen-like filamentous structures were observed around the pistil-like organ (arrowheads). Bar, 1 mm. (F) A developing panicle in the wild-type rice plant. Bar, 1 cm. (G) A mature floret of the wild-type rice. The floret was dissected to reveal internal organs. Bar, 5 mm. (H) Another rice terminal flower of a UBQ::FTL plant. Glumes were removed. Bar, 5 mm. (I) SEM image of the terminal flower in H. Bar, 5 mm. (J) A terminal tissue with many glumes that developed alternately. Several roots developed at the bases of this terminal tissue. Bar, 1 mm. (K) A terminal flower in glumes. An anther-like structure protruded next to the pistil-like organ (arrowhead). Bar, 1 mm. (L) Schematic presentation of UBQ::FTL phenotypes. Rice leaves are represented as thin lines (sheaths) with attached diamond shapes (blades). Florets are shown as oval shapes. The wild-type (WT) rice panicle is composed of primary and secondary branches with several florets. The terminal floret of UBQ::FTL plants is shown as a floret with additional glumes (UBQ::FTL). For simplification, axillary buds with terminal tissues are not shown in UBQ::FTL plants.

Figure 5

FT-like gene expression is controlled by SE5 and SE1. (A) Diurnal mRNA expression of three FT-like genes in wild-type (WT) and se5 plants under LD and SD conditions. RNA samples were obtained from 28-day-old plants (partly, the same RNA samples were used with those in Fig. 3B). Quantitative RT-PCR was performed twice to get average values. The normalized data are plotted. One time-point sample, for 18:00, is missing in se5 under SD conditions. Similar results were obtained with RNA samples harvested from 20-day-old plants. Different scales are used between the left and right graphs because of the differences in expression levels. (B) Diurnal mRNA expression of three FT-like genes under SD conditions in se1. RNA samples were obtained from 30-day-old plants. Quantitative RT-PCR was performed two to four times to get average values and standard deviations. The normalized data are plotted. (C) mRNA expression of three FT-like genes under LD conditions in se1 at different developmental stages by quantitative RT-PCR. Leaves were harvested at 09:30 in the morning every 10 d. RT-PCRs were performed several times to get averages and standard deviations. Representative data are shown from four, two, and two independent experiments on Hd3a, RFT1, and FTL expression, respectively. (D) mRNA expression of Hd3a in se1 and se1se5 plants under LD conditions at different developmental stages. Leaves were harvested at 10:30 every 10 d. Representative data from two independent experiments.

Figure 6

Model for day-length measurement in the photoperiodic control of flowering in rice. (A) In our model, the interaction between phytochrome signaling and the SE1 transcription factor makes SE1 a repressor of FT-like gene expression. In contrast, SE1 protein alone functions as an activator of FT-like gene expression. The interaction levels are higher under LD conditions than under SD conditions, as shown in B. The molecular basis of the interaction could be the formation of a transcriptional complex, because phytochrome is a nuclear factor. Phosphorylation of SE1 protein by phytochromes is an alternative. Gene X is postulated as a transcription factor that has a similar function to SE1, because mRNA expression of FT-like genes was detected under SD conditions even in the se1 mutant. (B) The amount of the active Pfr form of phytochromes is stable in the daytime and may gradually decrease with dark reversion. On the other hand, SE1 mRNA is nocturnally expressed by circadian clocks, and SE1 protein may exist mainly at a certain phase of subjective night. Phase setting of circadian clocks is determined by both light-on and light-off signals, whereas the decrease in levels of the Pfr form starts with the light-off signal. Thus, the timing of the coincidence of the Pfr form and the SE1 protein may differ among photoperiods. When the coincidence occurs, the interaction between Pfr and SE1 may make a repressor of FT-like genes. Without the coincidence, SE1 protein may function as an activator of FT-like genes to promote flowering in rice. Possible interactions between phytochrome and other factors than SE1 are omitted from this panel to focus on the relationship between SE1 and phytochrome.