The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.
Living organisms adapt to the environment by anticipating seasonal changes through day-length measurement, as seen in reproductive development in birds and mammals, diapause (a state of dormancy) in insects, and tuberization and flowering responses in higher plants (Lumsden & Millar 1998). This process of photoperiodism represents one of the most complex examples of the interaction between organisms and their environment. Flowering in higher plants is probably the most extensively studied photoperiodic response as it is so economically important in agriculture and horticulture.
Analyses of Arabidopsis photoreceptor mutants have revealed relatively small contributions of phytochromes in the photoperiodic control of flowering (Coupland 1998; Whitelam et al. 1998). phyA mutants of Arabidopsis flower later than the wild type under long-day conditions with a far-red enriched light source (Johnson et al. 1994), while in addition to PHYB, light-stable phytochromes regulate flowering in response to light quality in Arabidopsis (Simpson et al. 1999). In contrast, CRY2, a blue-light photoreceptor of Arabidopsis, plays an important role in the photoperiodic control of flowering (Guo et al. 1998). Therefore the relative importance of specific photoreceptors for light perception in photoperiodism may differ among plant species. Genetic analyses in Arabidopsis have also revealed a network of multiple loci regulating flowering time, and have allowed the dissection of the floral initiation process into genetically distinct signal transduction pathways (Koornneef et al. 1998; Piñeiro & Coupland 1998; Simpson et al. 1999). In these pathways, only floral induction under short-day conditions in the gibberellic acid (GA)-mediated pathway may be related to photoperiodic responses in short-day plants, although in most short-day plants GA application cannot promote floral initiation in non-inductive photoperiods (Thomas & Vince-Prue 1997). In addition, no pathway regulating floral inhibition under long-day conditions has been identified in Arabidopsis. Therefore these pathways are not sufficient to explain the photoperiodic response of short-day plants. Furthermore, physiological studies have suggested that the timekeeping mechanism of short-day plants in photoperiodism differs from that of long-day plants (Thomas 1998).
In this work we used a photoperiod-insensitive mutant photoperiodic sensitivity 5 (se5) (Yokoo & Okuno 1993) to investigate the photoperiodic control of flowering in rice, a facultative short-day plant. Cloning of the SE5 gene has revealed the phytochromes to be major photoreceptors for the photoperiodic control of flowering in rice.
The se5 mutant is photoperiod-insensitive
The recessive se5 mutant of rice has a pale, early flowering phenotype and is insensitive to extended light treatment with fluorescent lamps (Yokoo & Okuno 1993). To further analyse the photoperiodic control of flowering in the se5 mutant, plants were grown under three different photoperiods. Under long-day conditions (LD: 14 h light/10 h dark), se5 flowered more than 50 days earlier than the wild type (Fig. 1a,b). Under short-day conditions (SD: 10 h light/14 h dark), se5 plants flowered ≈15 days earlier than the wild-type plants (Fig. 1b). Thus the se5 plants flowered approximately 1 week earlier under LD than SD. This may be due to slower plant growth under SD; leaf emergence was slightly delayed under SD relative to LD (data not shown). Under continuous light conditions (24L: 24 h light), se5 plants flowered ≈46 days after sowing, while in the wild type the transition from vegetative to inflorescence meristems was not observed even at 120 days after sowing (Fig. 1b). On the other hand, the transition to inflorescence meristem was observed in the se5 plants ≈20 days after sowing (data not shown).
The size of the se5 plants on flowering was similar to that of the wild type under LD (Fig. 1a) and all other photoperiod conditions examined (data not shown). The rate of leaf emergence in se5 plants was also indistinguishable from that of the wild type, until flowering, under LD (Fig. 1c) and all other conditions. Leaf emergence reflects the rate of leaf primordia formation in rice (Itoh et al. 1998), which was apparently not affected by the se5 mutation. Therefore neither plant growth nor inflorescence development was altered by the se5 mutation. These results demonstrate that se5 is unable to delay flowering in response to LD and is therefore completely deficient in the photoperiodic control of flowering. Furthermore, the observation that se5 has an early flowering phenotype even under SD indicates that the SE5 gene actively inhibits floral induction under all the photoperiod conditions examined.
se5 lacks light responses and spectrophotometrically detectable phytochromes
In etiolated rice seedlings, elongation of the coleoptile has been shown to be regulated by phytochromes (Pjon & Furuya 1967). We used this response to assess whether se5 also affected light perception in de-etiolation. In the wild type, a single red light pulse resulted in ≈50% inhibition of coleoptile elongation compared with dark-grown seedlings, whereas a far-red light pulse following the red light pulse resulted in ≈25% inhibition (Fig. 2a). This 25% far-red-reversible inhibition may be attributed to phyB-type phytochromes (Quail et al. 1995; Whitelam et al. 1998). In addition, a single far-red pulse caused ≈25% inhibition in the wild type, a response probably mediated by phyA-type phytochromes (Quail et al. 1995; Whitelam et al. 1998). In contrast to the wild-type seedlings, se5 completely lacked responsiveness to any of the light pulses tested (Fig. 2a), indicating that light responses mediated by both phyA- and phyB-type phytochromes are missing in se5.
We next examined whether se5 had any effect on the content of spectrophotometrically detectable phytochrome. Crude extracts from etiolated rice seedlings exhibited a characteristic difference spectrum (Fig. 2b) due to the red/far-red reversible phototransformation of phytochromes, as shown by Pjon & Furuya (1968). However, in the se5 seedlings we were not able to detect any photoreversible phytochromes, even in 30-fold concentrated extracts (Fig. 2b), indicating that the se5 mutant contains less than 1% of the photoreversible phytochrome present in the wild type.
Growth of se5 seedlings under continuous light conditions
As phytochromes are involved in photomorphogenesis throughout the life cycle of higher plants (Quail et al. 1995; Whitelam et al. 1998), growth in continuous light conditions was examined for se5 seedlings (Fig. 3). Most plant parts examined were 20–30% longer in se5 than in the wild type in both red and far-red continuous light, whereas the difference under blue and white light was somewhat less. Lengths of second blades were not affected significantly under any light conditions tested and did not show clear differences between the wild type and se5. On the other hand, the second sheath of rice seedlings exhibited distinct responses under various light conditions, and significant differences were observed between the wild type and se5 in both red and far-red lights. These results are consistent with the idea that se5 is deficient in phytochromes. In addition, the results support the conclusion that both phyA- and phyB-mediated photomorphogenesis is affected in se5, as in other species these photoreceptors mediate responses to continuous far-red and red light, respectively (Whitelam et al. 1998). Under continuous white light there were smaller differences between the wild type and se5, except in coleoptiles (Fig. 3d), consistent with the observation that leaf emergence was normal in se5 under white light (Fig. 1a,c).
Molecular characterization of the SE5 gene
Because se5 was deficient in both light responses and spectrally active phytochromes, we examined the possibility that se5 might be a phytochrome chromophore-deficient mutant. We first sought a rice homologue of the recently isolated Arabidopsis HY1 gene, which encodes a plastid heme oxygenase known to function in chromophore biosynthesis (Davis et al. 1999; Muramoto et al. 1999). We found one rice EST clone (accession no. C28969) with a high similarity to HY1 which we temporarily designated OsHY1. Using rice recombinant inbred lines, this gene was mapped at ≈80 cm on chromosome 6 (data not shown), close to the map position previously reported for se5 (Yokoo & Okuno 1993), suggesting that the SE5 gene may be OsHY1. We therefore examined whether se5 might contain a mutation in OsHY1.
This analysis revealed a 1 bp deletion in the first exon of this gene in se5 (Fig. 4a), which caused a frame-shift and resulted in a premature stop codon. In the wild type, OsHY1 mRNA was slightly downregulated in light-grown relative to dark-grown seedlings. However, in se5 OsHY1 expression was reduced to ≈10% of the wild-type level and showed no light regulation (Fig. 4b). This reduction in mRNA accumulation may be caused by the premature termination codon created by the mutation, as it is known that the level of unproductive mRNA is reduced in eukaryotes, including plants (Hentze & Kulozik 1999). Thus it is likely that the one-base deletion of OsHY1 in se5 results in a loss of gene function.
To confirm that the SE5 gene was in fact OsHY1, we performed a complementation test by introducing a OsHY1 cDNA under the control of the CaMV 35S promoter into the se5 mutant by Agrobacterium-mediated transformation (Hiei et al. 1994). Progenies of four independent transgenic se5 plants carrying the 35S:OsHY1 construct were analysed for flowering time (Fig. 4d). In each of the four lines, progeny plants carrying the transgene exhibited clear photoperiodic responses and flowered at times similar to those of the wild-type plants. Segregants lacking the transgene flowered much earlier. Therefore we conclude that SE5 encodes OsHY1, a rice homologue of Arabidopsis HY1.
It has been shown that exogenously supplied biliverdin IXα (BV), a product synthesized by the HY1 heme oxygenase, can restore a wild-type light-grown phenotype in Arabidopsis hy1 (Parks & Quail 1991). Therefore we tested whether the se5 phenotype might be rescued by BV contained in the germination medium, by examining inhibition of coleoptile elongation by red light pulses. No restoration of coleoptile responses was seen (data not shown). The ineffectiveness of BV could have resulted from uptake problems; a similar observation was made for a chromophore-deficient pcd1 mutant of pea (Terry 1997; Weller et al. 1996).
Sequence comparison of SE5 and HY1 genes
SE5 encodes a single open reading frame of 289 amino acids (Fig. 4b). Genomic DNA blot analysis showed that SE5 is a single-copy gene in the rice genome (data not shown). The deduced amino acid sequence contains a putative transit peptide to the plastid of 65 amino acids at the N-terminus. This region is rich in alanine (15 aa/65 aa) and serine (10 aa/65 aa), but is quite distinct from the serine-rich transit peptide of the HY1 gene product. The difference in the transit peptide between SE5 and HY1 (Fig. 4b) may reflect distinct mechanisms for protein localization to the plastid in rice and Arabidopsis. The rest of SE5 shows ≈72% identity with HY1 (Fig. 4b), which strongly suggests that the SE5 gene product can function as a heme oxygenase (Muramoto et al. 1999). Since the positions of the first and second introns are conserved between SE5 and HY1 (Fig. 4b), these genes may have originated from the same ancestor gene.
Phytochromes and photoperiodism
We have shown that a deficiency in phytochrome chromophore results in complete loss of the ability of non-inductive long-day conditions to delay flowering in rice. This clearly shows that phytochromes play a major role in the photoperiodic control of flowering in rice. The early flowering phenotype of se5 under long-day conditions (even under 24L) led us to conclude that the se5 mutant is effectively null for photoperiodic response. As se5 can grow and develop leaves in the same way as the wild type (Fig. 1a,c), it is clear that the mutant must contain some functional photoreceptors active under white light. However, the low level of phytochromes in se5 is clearly insufficient to transmit light signals for day-length measurement.
It is still unclear which phytochrome species contributes to the delay in flowering under long-day conditions: multiple phytochromes are known in rice and se5 is likely to affect the levels of them all (Dehesh et al. 1991; Kay et al. 1989; Tahir et al. 1998). Because the wild-type SE5 gene can inhibit flowering under 24L, light-stable phytochromes are likely to be involved in floral inhibition of rice (Fig. 1b).
Like the se5 mutants of rice, the line of the SDP sorghum is early flowering and exhibits a severely reduced photoperiodic response. This phenotype results from a deficiency in phyB (Childs et al. 1997). However, plants retained a 2-week delay in floral initiation under 24L compared with SD (Childs et al. 1995). This residual photoperiodic response in the sorghum line may be attributed to other light-stable phytochromes such as PHYC. The fact that the photoperiodic response is completely absent in se5 probably reflects a severe reduction in the level of all phytochromes in this mutant.
Light-stable phytochromes also have a clear role in the inhibition of flowering in LDP (Thomas 1999). A phyB-deficient mutant of pea flowered earlier than the wild type in short-days but not in long-days (Weller & Reid 1993), while in Arabidopsis, phyB mutants also flower early, an effect more pronounced in short-days than in long-days. However, both pea and Arabidopsis mutants retain some photoperiodic responsiveness (Goto et al. 1991; Mockler et al. 1999; Weller & Reid 1993). Analyses of other light-stable phytochrome mutants of Arabidopsis, phyD and phyE, also support this conclusion (Whitelam et al. 1998). In an obligate photoperiodic potato species which tuberizes only in short-days, transgenic plants expressing antisense PHYB mRNA are able to tuberize in long-days as well as in short-days (Jackson et al. 1996). These results also indicate important roles of light-stable phytochromes in the photoperiodic response. Interestingly, phyB mutants of a near day-neutral plant, Nicotinia plumbaginifolia, exhibited late flowering, indicating that light-stable phytochromes can promote flowering in this species (Hudson & Smith 1998).
In contrast to phyB, light-labile phyA is thought to promote flowering in some LDP species. The phyA-deficient mutant of pea, fun1, flowers later than the wild type in long-days but not in short-days, resulting in reduced photoperiodic responses (Weller et al. 1997). phyA also promotes flowering in Arabidopsis: the phyA mutant flowers significantly later than the wild type in response to day-length extensions with a far-red-enriched white light (Johnson et al. 1994). So far no phyA mutants have been isolated in SDP. Our results in rice have shown although most photoreversible phyA was lost in se5 (Fig. 2b), this did not confer a late-flowering phenotype under any of the conditions examined (Fig. 1b). This could suggest that phyA may not play an important role in the photoperiodic control of flowering in rice. Alternatively, phyA effects on flowering may be dependent on the action of other phytochromes, and in the absence of these phytochromes the effect of loss of phyA may not be seen.
se5 may be homologous to hy1 in Arabidopsis
Several lines of evidence indicate that SE5 gene of rice is a counterpart of the Arabidopsis HY1 gene. First, the putative heme oxygenase domain encoded by SE5 shares more than 70% identity with that of the HY1 product. SE5 is more similar to HY1 than to another HY1-like protein, AtHO2, in Arabidopsis (Davis et al. 1999). Conservation of intron positions between SE5 and HY1, and the existence of a putative transit peptide in SE5, also support this idea (Fig. 4b). Secondly, like hy1, the se5 mutant is deficient in phytochrome responses such as coleoptile responses by light pulses (Fig. 2a) and seedling growth under continuous red and far-red lights (Fig. 3a,b). In addition, se5 lacks photoreversible phytochromes in extracts (Fig. 2b). Thirdly, SE5 appears to be a single-copy gene, not a member of a gene family. Wild-type SE5 mRNA expression was slightly higher in dark-grown than in light-grown seedlings (Fig. 3c). On the other hand, in Arabidopsis HY1 expression is modestly light-regulated (Davis et al. 1999), suggesting distinct regulation of gene expression of the plastid heme oxygenase between rice and Arabidopsis.
Comparison between se5 and hy1 mutants
The Arabidopsis hy1 mutant is early flowering and photoperiod-sensitive. The number of rosette leaves on flowering is reduced under both day lengths, and rosette leaf number is higher under short-day than under long-day conditions (Goto et al. 1991). Furthermore, the number of days to flowering in hy1 is not different from the wild type in both long- and short-days (Goto et al. 1991), indicating later leaf emergence in hy1 than in the wild type. This effect of hy1 on leaf emergence makes the evaluation of photoperiodic responses of hy1 difficult. In contrast, leaf emergence of rice was not affected by the se5 mutation (Fig. 1c). The difference between rice se5 and Arabidopsis hy1 may reflect distinct physiological roles of phytochromes between rice and Arabidopsis in various phases of plant development. Recently, transgenic Arabidopsis plants overexpressing the mammalian biliverdin IXα reductase in plastids were shown to mimic hy1 phenotypes and retain floral responses to night-break treatments (Montgomery et al. 1999). Therefore phytochrome-chromophore deficiency may not be sufficient to lose photoperiodic responses in Arabidopsis. Photoperiodic responses retained in hy1 may be caused by other photoreceptors. One of the cryptochrome mutants, cry2, was shown to be allelic to an Arabidopsis late-flowering mutant, fha, which exhibited a severely reduced photoperiodic response (Guo et al. 1998).
Effects of the se5 mutation on photomorphogenesis in rice seedlings may also differ from those of hy1. Compared with the wild type, hypocotyl length in hy1 is almost three times longer in continuous far-red light, and about twice as long in continuous red light (Ahmad & Cashmore 1997; Parks & Quail 1993). No significant changes in elongation were seen in hy1 seedlings under blue light (Ahmad & Cashmore 1997). In contrast, elongation of se5 seedlings under red and far-red light was only 20–30% higher than in the wild type (Fig. 3a,b). Under white light, hy1 is around 2–2.5 times more elongated than the wild type (Koornneef et al. 1980; Parks & Quail 1993; Reed et al. 1993), while the growth of se5 seedlings did not differ significantly from the wild type under white light (Fig. 3d). Elongation of mesocotyl was not sufficient to be measured in either wild type or se5 under continuous light. These results suggest there may be differences in the relative importance of various photoreceptors for photomorphogenesis between these two species. Like hy1, significant elongation of seedlings under white light was reported in the pcd1 mutant of pea and yg2 mutant of tomato, which were shown to lack heme oxygenase activity and are likely to be phytochrome chromophore-deficient mutants (Terry & Kendrick 1996; Terry 1997; Weller et al. 1996). Therefore it is likely that the pea PCD1 and tomato YG2 genes may encode heme oxygenase genes homologous to SE5 and HY1.
Phytochromes are involved in many physiological phenomena such as photomorphogenesis, seed germination, chloroplast differentiation, pigment synthesis, and control of flowering time (Quail et al. 1995; Whitelam et al. 1998). Our results indicate that the photoperiodic control of flowering in rice is very sensitive to phytochrome deficiency. Therefore rice might be a good model for short-day plants to elucidate interaction between phytochromes and time-keeping mechanisms in the photoperiodic control of flowering. Recently, genetic analyses among rice cultivars and QTL analyses in rice have revealed multiple loci regulating floral initiation (Ichitani et al. 1997, 1998; Yamamoto et al. 1998; Yamamoto et al. 2000). It is of interest to examine the interaction of these genetic loci and SE5 in the regulation of flowering time. Isolation of phytochrome-specific mutants will be necessary in order to examine the individual contribution of phyA and B in the photoperiodic response of rice.
Plant material and growth conditions
The se5 mutant was isolated from Oryza sativa cv. Norin 8 by X-ray radiation mutagenesis (Yokoo & Okuno 1993). Plants were grown in climate chambers for flowering-time experiments with 24 h temperature cycles (12 h, 30°C for subjective day/12 h, 25°C for subjective night). The fluence rates of light were ≈300 μmol m−2 sec−1 (400–750 nm) under LD and SD, and ≈150 μmol m−2 sec−1 (400–750 nm) under 24L. The fluence rates of light were measured with a spectroradiometer (model LI-1800, Licor, Lincoln, NE). The heading date at which panicles emerged from the flag leaf was scored for dates of flowering. To examine leaf emergence, leaf numbers were scored when tips of a new leaf blade emerged from the sheath of the previous leaf.
For photomorphogenesis experiments, sterile rice seeds were imbibed on the medium at 4°C for 4 days and grown at 25°C for 8 days in lights of red, far-red, blue and white fluorescent lamps (National FL20SR-F, Toshiba FL20SFR-74, National FL20SB-F and Toshiba FL20SSBRN/18-A, respectively). For far-red light, coloured filters (red No. 22 and blue No. 72, Tokyo Butai Syoumei) were used to reduce extra red and blue lights. Light quality was confirmed using a spectroradiometer.
Coleoptile response by light pulses
The experiment was performed according to methods described previously (Pjon & Furuya 1967). Sterile seeds were imbibed in water for 1 day, transferred to the medium, then irradiated with far-red light for 15 min to transform the Pfr-active form of phytochromes in seeds to the Pr-inactive form. After 2 days in darkness, 15 min (red) and 30 min (far-red) light pulses were given, as shown in Fig. 2(a). The fluence rates were ≈14 μmol m−2 sec−1 for red (540–690 nm) and ≈6 μmol m−2 sec−1 for far-red (690–790 nm). At 3 days at 25°C after the pulse treatments, coleoptile lengths of ≈10 seedlings were measured for each treatment.
Spectrophotometric measurements of phytochrome
Measurement was done essentially using methods previously described (Pjon & Furuya 1968). Seven-day-old etiolated seedlings were homogenized on ice in 0.8 vol (0.8 ml/1.0 g) of a 100 mm potassium phosphate (pH 8.3) solution containing 5 mm Na2 EDTA and 28 μm 2-mercaptoethanol. The homogenate was filtered through two layers of cheesecloth and then centrifuged at 4°C to remove insoluble materials. The supernatant was recovered as a crude extract from the tissues. Solutes were precipitated by ammonium sulfate (230 mg ml−1), then redissolved in desired volumes of a 10 mm potassium phosphate (pH 7.8) solution containing 1 mm Na2 EDTA and 28 μm 2-mercaptoethanol for the concentration of extracts. The absorption difference spectra of phytochromes in the extract were measured with a Hitachi 557 spectrophotometer, as described by Saitou et al. (1999). All procedures were performed under a dim green safety light.
Cloning of SE5
Genomic clones corresponding to the EST (accession no. C28969) were isolated from a rice genomic library. The nucleotide sequence of OsHY1 in se5 was determined by direct sequencing of PCR products. OsHY1 cDNA clones were isolated from rice cDNA pools using a Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA, USA).
Complementation test by rice transformation
NotI and SacI sites at ends of the SE5 cDNA were created by PCR in vitro mutagenesis, and the sequence of the cDNA was verified. The NotI–SacI DNA fragment carrying SE5 cDNA was ligated into a binary vector downstream of the CaMV 35S promoter and was introduced into rice by Agrobacterium-mediated transformation (Hiei et al. 1994) and R1 seeds were obtained by self-pollination. Transmission of the transgenes to R1 progeny plants was confirmed by amplification of the DNA fragment specific to transgenes by PCR. Progeny plants were germinated in the climate chambers and grown until they flowered under either LD or SD conditions.
We thank Dr Takayuki Kohchi for amino acid sequence information on HY1, Daisuke Miki for the vector construction, Mika Nobuhara for preparation of transgenic rice plants, Drs Masaki Furuya and Junko Kyozuka for critical reading of the manuscript, and our colleagues for valuable discussions. This work was supported by a grant from the program Grants-in-Aid for Scientific Research on Priority Areas ‘Molecular Mechanisms Controlling Multicelluar Organization of Plants' of the Japanese Ministry of Education, Science, Culture, and Sports.