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Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice - PubMed

. 2005 Dec;17(12):3311-25.

doi: 10.1105/tpc.105.035899. Epub 2005 Nov 8.

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Free PMC article

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

Makoto Takano et al. Plant Cell. 2005 Dec.

Free PMC article

Abstract

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.

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Figures

Figure 1.
Figure 1.

Characterization of phyB and phyC Mutants. (A) Mutation sites found in the PHYB gene and insertion site of Tos17 in the PHYC gene are schematically depicted. Exons are represented as boxes in which closed parts are coding regions and open parts are 5′- and 3′-untranslated regions. The chromophore binding site is indicated by “#.” The horizontal arrow shows an orientation of the inserted Tos17 (gray bar). (B) Protein and RNA gel blot analyses of phyB mutant lines. Total proteins or RNA were extracted from 4-d-old etiolated seedlings. Top three lanes: 50 μg each of proteins were served for the detection of PHYA by the monoclonal anti-rye PHYA antibody (mAR07) or PHYB and PHYC by the polyclonal antibody raised against C-halves of PHYB and PHYC proteins, respectively. Bottom two lanes: each lane was loaded with 10 μg of total RNA for the detection of PHYB or PHYC transcripts. Nipponbare (Np) and Norin 8 (N8) were used as controls. (C) Dilution series of protein extract from Nipponbare were compared with the protein extract from phyB-1 for the PHYC level. (D) PHYA, PHYB, and PHYC proteins were detected by protein gel blotting of extracts from 4-d-old etiolated seedlings from Nipponbare (WT), phyA-2 (phyA), phyB-1 (phyB), and phyC-1 (phyC) mutants. (E) Immunoblot detection of PHYC proteins in Nipponbare (WT), phyA-2 (phyA), phyB-1 (phyB), and phyA-2 phyB-1 (phyAphyB) mutants. Four-day-old etiolated seedlings were used for the extraction of proteins. Each lane was loaded with 50 μg of protein for the detection of PHYC protein.

Figure 2.
Figure 2.

Nine-Day-Old Seedlings of Wild-Type and Phytochrome Mutants Grown under Rc or FRc. Seedlings were grown in darkness (A) or under Rc (B) or FRc (C) for 9 d at 28°C. The fluence rates of Rc and FRc are 15 μmol photons m−2 s−1. Two seedlings of Nipponbare (wild type) grown in dark are in (A). In (B) and (C), wild-type and single and double phytochrome mutants are aligned for comparison from left to right: wild type, phyA, phyB, phyC, phyA phyC, phyB phyC, and phyA phyB. White and yellow arrows indicate apexes of coleoptiles and first leaves, respectively. Red arrows indicate second nodes. All pictures are the same magnitude. Bars = 10 mm.

Figure 3.
Figure 3.

Lengths of Different Tissues of the Nine-Day-Old Seedlings Represented in Figure 2. Coleoptile lengths (A), lengths of first leaves (B), and lengths of second internodes (C) of 9-d-old seedlings are shown for wild-type and phytochrome mutants grown in the dark (black bars) or under Rc (gray bars) or FRc (white bars) as in Figure 2. The means ±

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were obtained from 20 to ∼50 seedlings.

Figure 4.
Figure 4.

Day Dependency of R Light Effect in Photoinhibition of Coleoptile Elongation. (A) Coleoptile lengths of Nipponbare (open bars) and phyB-1 seedlings (shaded bars) grown in different R light treatment conditions shown in (B). Bars represent relative values based on the average coleoptile lengths of dark-grown seedlings (condition 8). Overlaid red curve shows the growth curve of the coleoptile of Nipponbare seedlings in the complete darkness at 28°C. The means ±

se

obtained from 30 seedlings were plotted. (B) Schematic drawing of experimental timetable for exposure to Rc. Except for condition 8 (in complete darkness for 7 d) and condition 9 (under Rc for 7 d), each set was exposed to Rc (15 μmol photons m−2 s−1) for 1 d in a designated timing.

Figure 5.
Figure 5.

Second Leaf Blade Declination Induced by Bc and Wc. (A) and (B) Seedlings were grown under Bc (A) or Wc (B) for 9 d at 28°C. The fluence rates of Bc and Wc are 15 and 40 μmol photons m−2 s−1, respectively. Wild-type and single and double phytochrome mutants are aligned for comparison from left to right: wild type, phyA, phyB, phyC, phyA phyC, phyB phyC, and phyA phyB. Yellow arrows indicate lamina joints of second leaves. The two pictures are the same magnitude, and scale bars at left sides are 10 mm. (C) Declination angles are measured from the pictures and shown for wild-type and phytochrome mutants grown under Bc (shaded bars) or Wc (open bars). The means ±

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were obtained from 20 to ∼50 seedlings.

Figure 6.
Figure 6.

Induction of Lhcb Gene Expression by R and/or FR Pulses in Etiolated Rice Seedlings. Rice seedlings from Nipponbare (WT) and phytochrome single (phyA, phyB, and phyC) and double (phyA phyC, phyB phyC, and phyA phyB) mutants were grown in complete darkness for 7 d (D) and then treated with a single Rp (R), a FRp immediately after an Rp (R/FR), a train of Rp-FRp-Rp (R/FR/R), or a single FRp (FR). Seedlings were harvested 3 h after the pulse irradiation, and Lhcb gene expression was analyzed by RNA gel blot hybridization. rRNA was stained by methylene blue with the blot of wild type as a quantity control.

Figure 7.
Figure 7.

Induction of Lhcb and RbcS Genes by FRc or Rc in Wild-Type and Phytochrome Mutant Seedlings. Nipponbare (WT) and phytochrome single (phyA, phyB, and phyC) and double (AB, phyA phyB; AC, phyA phyC; BC, phyB phyC) mutants were grown in the dark (D) or under FRc (FR in [A]) or Rc (R in [B]) for 4 d. Lhcb and RbcS gene expression was analyzed by RNA gel blot hybridization. rRNA was stained by methylene blue with the same blot as a quantity control.

Figure 8.
Figure 8.

Comparison of Flowering Times between Wild-Type and phyB Mutants under Natural Daylengths and SD Conditions. (A) and (B) Nipponbare (WT) and phytochrome single (phyA, phyB, and phyC) and double (phyA phyC, phyB phyC, and phyA phyB) mutants were grown in the paddy field (A) or in a growth chamber set as SD (10L/14D; [B], open bars) or LD (14L/10D; [B], shaded bars), and their flowering times were measured (natural daylength). (C) Wild-type (open bars) and phyB mutants (hatched bars) were grown in a growth chamber set as SD (normal, 10L/14D; severe, 8L/16D) or SD (10L/14D) plus EDO-FR conditions. The means ±

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obtained from 20 plants are displayed.

Figure 9.
Figure 9.

Two-Way Table of Flowering Time for Single and Double Phytochrome Mutants under SD and LD Conditions in Rice and Arabidopsis. Value of each cell represents the effect of a single or a double mutation indicated by a combination of the corresponding row (left) and column (top). Combinations of the same mutation in row and column indicate the results for the single mutants (e.g., a cell with row phyA and column phyA indicates a value from phyA single mutants). To facilitate the comparison of data in one row, results from double mutants are duplicated in the table (e.g., phyA phyB and phyB phyA are the same data). Flowering time of each mutant is compared with that of the wild type in the same photoperiod. Figures in the cells are relative values of flowering time based on the wild type and visualized by arrows at the left. Horizontal arrows, same as the wild type; downward arrows, earlier than the wild type; upward arrows, later than the wild type. The number of arrows represents the extent of the effect. The values of Arabidopsis are calculated from the data reported by Reed et al. (1994), Neff and Chory (1998), and Monte et al. (2003). Photoperiods with the same effects on the flowering of each plant (LD for rice and SD for Arabidopsis are suppressive, and SD for rice and LD for Arabidopsis are inductive) are aligned.

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