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.

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.

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.

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.

In order to elucidate the function of cryptochromes (cry) in rice, we have characterized all rice CRY genes, including OsCRY1a, OsCRY1b and OsCRY2 . Our studies revealed that OsCRY1 genes were mainly expressed in the green plant tissue, while OsCRY2 gene expression was high in the coleoptile, flower and callus. Light treatment affected neither the expression of any of the OsCRY genes nor the stability of their transcripts. However, it was found that Oscry2 protein was negatively regulated by blue light. Moreover, the level of Oscry2 protein also decreased upon red light treatment, and this red light-dependent degradation was shown to be mediated by phytochrome B. Overexpression of OsCRY1 genes resulted in an increased responsiveness to blue light when measuring coleoptile growth inhibition. Moreover, growth of leaf sheaths and blades was also repressed more in OsCRY1 overexpressers than in wild type (WT) under blue light conditions. These results suggest that Oscry1s are responsible for regulating blue light-mediated de-etiolation in rice. In addition, OsCRY2 antisense transgenic rice flowered later than WT under both long-day and short-day conditions, indicating that Oscry2 is involved in the promotion of flowering time in rice.