Tomoko Shinomura

Distinct and Cooperative Functions of Phytochromes A, B, and C in the Control of Deetiolation and Flowering in Rice

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.

Isolation and characterization of rice phytochrome A mutants

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception—far-red light–dependent high irradiance response and very low fluence response—and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.

Suppression of the Floral Activator Hd3a Is the Principal Cause of the Night Break Effect in Rice

A short exposure to light in the middle of the night causes inhibition of flowering in short-day plants. This phenomenon is called night break (NB) and has been used extensively as a tool to study the photoperiodic control of flowering for many years. However, at the molecular level, very little is known about this phenomenon. In rice (Oryza sativa), 10 min of light exposure in the middle of a 14-h night caused a clear delay in flowering. A single NB strongly suppressed the mRNA of Hd3a, a homolog of Arabidopsis thaliana FLOWERING LOCUS T (FT), whereas the mRNAs of OsGI and Hd1 were not affected. The NB effect on Hd3a mRNA was maximal in the middle of the 14-h night. The phyB mutation abolished the NB effect on flowering and Hd3a mRNA, indicating that the NB effect was mediated by phytochrome B. Because expression of the other FT-like genes was very low and not appreciably affected by NB, our results strongly suggest that the suppression of Hd3a mRNA is the principal cause of the NB effect on flowering in rice.

The Novel MYB Protein EARLY-PHYTOCHROME-RESPONSIVE1 Is a Component of a Slave Circadian Oscillator in Arabidopsis

Using fluorescent differential display, we identified, from ∼8000 displayed bands, a DNA fragment showing rapid induction in response to red light irradiation. This EARLY-PHYTOCHROME-RESPONSIVE1 gene (EPR1) encodes a novel nucleus-localized MYB protein harboring a single MYB domain that is highly similar to the circadian oscillator proteins CCA1 and LHY. EPR1 is regulated by both phytochrome A and phytochrome B, and the red-light induction of EPR1 is not inhibited by cycloheximide, demonstrating that EPR1 represents a primary phytochrome-responsive gene. Our results show that EPR1 overexpression results in enhanced far-red light–induced cotyledon opening and delayed flowering. In wild-type Arabidopsis plants grown in continuous light, the EPR1 transcript exhibits circadian rhythmicity similar to that of CCA1 and LHY. Moreover, EPR1 suppresses its own expression, suggesting that this protein is part of a regulatory feedback loop. Constitutive expression of CCA1 and LHY results in the loss of EPR1 rhythmicity, whereas increased levels of EPR1 have no effect on the central oscillator. We propose that EPR1 is a component of a slave oscillator that contributes to the refinement of output pathways, ultimately mediating the correct oscillatory behavior of target genes.

Phytochrome B regulates Heading date 1 (Hd1)-mediated expression of rice florigen Hd3a and critical day length in rice

Many plants require circadian clock and light information for the photoperiodic control of flowering. In Arabidopsis, a long-day plant (LDP), flowering is triggered by the circadian clock-controlled expression of CONSTANS (CO) and light stabilization of the CO protein to induce FT (FLOWERING LOCUS T). In rice, a short-day plant (SDP), the CO ortholog Heading date 1 (Hd1) regulates FT ortholog Hd3a, but regulation of Hd3a by Hd1 differs from that in Arabidopsis. Here, we report that phytochrome B (phyB)-mediated suppression of Hd3a is a primary cause of long-day suppression of flowering in rice, based on the three complementary discoveries. First, overexpression of Hd1 causes a delay in flowering under SD conditions and this effect requires phyB, suggesting that light modulates Hd1 control of Hd3a transcription. Second, a single extension of day length decreases Hd3a expression proportionately with the length of daylight. Third, Hd1 protein levels in Hd1-overexpressing plants are not altered in the presence of light. These results also suggest that phyB-mediated suppression of Hd3a expression is a component of the molecular mechanism for critical day length in rice.

Phytochromes are the sole photoreceptors for perceiving red/far-red light in rice

Phytochromes are believed to be solely responsible for red and far-red light perception, but this has never been definitively tested. To directly address this hypothesis, a phytochrome triple mutant (phyAphyBphyC) was generated in rice (Oryza sativa L. cv. Nipponbare) and its responses to red and far-red light were monitored. Since rice only has three phytochrome genes (PHYA, PHYB and PHYC), this mutant is completely lacking any phytochrome. Rice seedlings grown in the dark develop long coleoptiles while undergoing regular circumnutation. The phytochrome triple mutants also show this characteristic skotomorphogenesis, even under continuous red or far-red light. The morphology of the triple mutant seedlings grown under red or far-red light appears completely the same as etiolated seedlings, and they show no expression of the light-induced genes. This is direct evidence demonstrating that phytochromes are the sole photoreceptors for perceiving red and far-red light, at least during rice seedling establishment. Furthermore, the shape of the triple mutant plants was dramatically altered. Most remarkably, triple mutants extend their internodes even during the vegetative growth stage, which is a time during which wild-type rice plants never elongate their internodes. The triple mutants also flowered very early under long day conditions and set very few seeds due to incomplete male sterility. These data indicate that phytochromes play an important role in maximizing photosynthetic abilities during the vegetative growth stage in rice.

Phytochrome regulation of seed germination.

Seed germination of many plant species is influenced by light. Of the various photoreceptor systems, phytochrome plays an especially important role in seed germination. The existence of at least five phytochrome genes has led to the proposal that different members of the family have different roles in the photoregulation of seed germination. Physiological analysis of seed germination ofArabidopsis thaliana (L.) Heynh. with phytochrome-deficient mutants showed for the first time that phytochrome A and phytochrome B modulate the timing of seed germination in distinct actions. Phytochrome A photo-irreversibly triggers the photoinduction of seed germination after irradiation with extremely low fluence light in a wide range of wavelengths, from UV-A, to visible, to far-red. In contrast, phytochrome B mediates the well-characterized photoreversible reaction, responding to red and far-red light of fluences four orders of magnitude higher than those to which PhyA responds. Wild plants, such asA. thaliana, survive under ground as dormant seeds for long periods, and the timing of seed germination is crucial for optimizing growth and reproduction. It therefore seems reasonable for plants to possess at least two different physiological systems for sensing the light environment over a wide spectral range with exquisite sensitivity of different phytochromes. This redundancy seems to enhance plant survival in a fluctuating environment.

Fluence and Wavelength Requirements for Arabidopsis CAB Gene Induction by Different Phytochromes

The roles of different phytochromes have been investigated in the photoinduction of several chlorophyll a/b-binding protein genes (CAB) of Arabidopsis thaliana. Etiolated seedlings of the wild type, a phytochrome A (PhyA) null mutant (phyA), a phytochrome B (PhyB) null mutant (phyB), and a phyA/phyB double mutant were exposed to monochromatic light to address the questions of the fluence and wavelength requirements for CAB induction by different phytochromes. In the wild type and the phyB mutant, PhyA photoirreversibly induced CAB expression upon irradiation with very-low-fluence light of 350 to 750 nm. In contrast, using the phyA mutant, PhyB photoreversibly induced CAB expression with low-fluence red light. The threshold fluences of red light for PhyA- and PhyB-specific induction were about 10 nmol m-2 and 10 [mu]mol m-2, respectively. In addition, CAB expression was photoreversibly induced with low-fluence red light in the phyA/phyB double mutant, revealing that another phytochrome(s) (PhyX) regulated CAB expression in a manner similar to PhyB. These data suggest that plants utilize different phytochromes to perceive light of varying wave-lengths and fluence, and begin to explain how plants respond so exquisitely to changing light in their environment.

Structural requirement of bilin chromophore for the photosensory specificity of phytochromes A and B

Phytochromes are an important class of chromoproteins that regulate many cellular and developmental responses to light in plants. The model plant species Arabidopsis thaliana possesses five phytochromes, which mediate distinct and overlapping responses to light. Photobiological analyses have established that, under continuous irradiation, phytochrome A is primarily responsible for plants sensitivity to far-red light, whereas the other phytochromes respond mainly to red light. The present study reports that the far-red light sensitivity of phytochrome A depends on the structure of the linear tetrapyrrole (bilin) prosthetic group. By reconstitution of holophytochrome in vivo through feeding various synthetic bilins to chromophore-deficient mutants of Arabidopsis, the requirement for a double bond on the bilin D-ring for rescuing phytochrome A function has been established. In contrast, we show that phytochrome B function can be rescued with various bilin analogs with saturated D-ring substituents.

Phytochrome-mediated inhibition of coleoptile growth in rice : Age-dependency and action spectra

Phytochrome has been shown to be the major photoreceptor involved in the photo‐inhibition of coleoptile growth in Japonica‐type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age‐dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photo‐inhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo‐inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low‐fluence range (101–103 μmol m−2) of R and was far‐red‐light (FR)‐reversible; the photo‐inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very‐low‐fluence range (10−2–100 μmol m−2) and was FR‐irreversible. The action spectra (350–800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB‐mediated low‐fluence response and the phyA‐mediated very‐low‐fluence response have a major peak at 650 nm and a minor peak at 400 nm.