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(American Journal of Botany. 2009;96:1319-1336.) doi: 10.3732/ajb.0800340 © 2009 Botanical Society of America, Inc. |
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Physiology and Biochemistry |
2 Section of Plant Biology, College of Biological Sciences, One Shields Avenue, University of California, Davis, California 95616 USA 3 Department of Plant Sciences, Center for Plant Diversity, UC Davis Herbarium, College of Agriculture and Environmental Sciences, 2041 Wickson Hall, University of California, Davis USA
Received for publication 7 October 2008. Accepted for publication 11 February 2009.
ABSTRACT
To investigate the role of distinct phytochrome pools in photoperiodic timekeeping, we characterized four phytochrome genes in the short-day plant Pharbitis nil. Each PHY gene had different photosensory properties and sensitivity to night break that inhibits flowering. During extended dark periods, PHYE, PHYB, and PHYC mRNA accumulation exhibited a circadian rhythmicity indicative of control by an endogenous clock. Phylogenetic analysis recovered four clades of angiosperm phytochrome genes, phyA, phyB, phyC, and phyE. All except the phyE clade included sequences from both monocots and eudicots. In addition, phyA is sister to phyC and phyE sister to phyB, with gymnosperm sequences sister to either the phyA-phyC clade or to the phyB-phyE clade. These results suggest that a single duplication occurred in an ancestral seed plant before the divergence of extant gymnosperms from angiosperms and that two subsequent duplications occurred in an ancestral angiosperm before the divergence of monocots from eudicots. Thus in P. nil, a multigene family with different patterns of mRNA abundance in light and darkness contributes to the total phytochrome pool: one pool is light labile (phyA), whereas the other is light stable (phyB and phyE). In addition, PHYC mRNA represents a third phytochrome pool with intermediate photosensory properties.
Key Words: circadian rhythm • Convolvulaceae • endogenous clock • flowering • night break • Pharbitis nil • photoperiodism • phylogeny • phytochrome • short-day plant
In photoperiodic flowering plants, the transition from vegetative to reproductive growth is controlled by photoperiod or, in more precise wording, by the relative length of the day and night in a 24-h cycle (Garner and Allard, 1920
). Photoperiodic flowering is controlled by inductive processes that occur in vegetative tissues, either the seedling cotyledon or mature leaf, and is mediated by phytochrome, the red–far-red photoreceptor that controls plant growth and development. These phytochrome-mediated processes result in the synthesis and translocation of a floral stimulus, or flowering hormone, to the shoot apex where flowering occurs. The photoperiodic control of flowering has been a topic of intense research for nearly a century and has led to a number of fundamental and foundational discoveries about light-regulation of development, including the discovery of phytochrome itself.
Research on photoperiodism and flowering, as well as on the identification and activity of phytochrome as a light sensor, has led to the formulation of a general model depicting photoperiodic flowering as a series of sequential partial processes that couple stimulus (darkness of a critical duration) to response (flowering), as depicted in the model for photoperiodic flowering shown in Fig. 1. According to this classic model, flowering occurs when a time-measuring process in the leaf, initially sensed and subsequently mediated by phytochrome, leads to the synthesis of a flower-promoting stimulus that is translocated to the shoot where it evokes the floral transition of the meristems, resulting in flower bud differentiation (after Lang, 1965). After 100 years of research, this sequential model of photoperiodic flowering is still relevant today. The model also illustrates what has been termed the "central mystery" of photoperiodic flowering (Evans, 1969), namely, how with a common beginning (phytochrome mediation of the initial photoperiodic reactions in all plants) and a common ending (production of a translocatable flowering hormone), the intermediate reactions require darkness in short-day plants (SDPs) and light in long-day plants (LDPs). The "central mystery" has been largely resolved in Arabidopsis thaliana Heyn. and Oryza sativa L. (rice), two model systems that have been studied extensively with respect to the regulatory genes that control photoperiodic flowering, as discussed later.
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Numerous detailed physiological studies have characterized photoperiodically controlled flowering and the intersecting role of an endogenous timekeeping mechanism (Bünning, 1936; Hamner, 1958; Lang, 1965; Vince-Prue, 1975, 1986; Zeevaart, 1976; Heide, 1977; Bernier et al., 1981; Salisbury, 1982; Bernier, 1988; Lumsden, 1991; Evans, 1969, 1993; O’Neill, 1992, 1993; McClung, 2006). More recent reviews reflect the many major advances made using model genetic systems such as Arabidopsis and rice (Sugiyama et al., 2001; Mouradov et al., 2002; Perilleux and Bernier, 2002; Yanovsky and Kay, 2002; Izawa et al., 2003; Sullivan and Deng, 2003; Hayama and Coupland, 2004; Ausín et al., 2005; Franklin et al., 2005; Corbesier and Coupland, 2006; Imaizumi and Kay, 2006; Thomas, 2006; Zeevaart, 2006; Kobayashi and Weigel, 2008). However, in terms of photoperiodic response, the field of flowering is characterized by complexity and diversity rather than by simplicity and uniformity, with short-day plants (SDPs), long-day plants (LDPs), and various combinations of these types, as well as SDPs that differ from one another, and LDPs that do the same. Thus, the tremendous diversity of responses of plant species in their ecologically adapted environments indicates that many species are idiosyncratic in their photoperiodic response.
Although there are many variations on the photoperiodic theme, the physiology of floral induction has been most extensively studied in species that have an obligate, qualitative sensitivity to photoperiod. The best characterized are the obligate, qualitative SDP and LDP systems. Based on a 24-h period, a SDP will be induced to flower when the dark period exceeds a critical night length (CNL), whereas a LDP will flower when the dark period is less than the CNL. In contrast, most of the molecular genetic work that has illuminated the "central mystery" of flowering has been done on facultative, quantitative photoperiodic response types, such as the LDP Arabidopsis and the SDP rice. The major advances made in our understanding of the mechanism of photoperiodic flowering in these two model systems has been compared in recent reviews (Izawa et al., 2003; Hayama and Coupland, 2004).
It is now widely understood that Arabidopsis and rice share similar flowering time genes that integrate signals from the circadian clock and the photoreceptors to either promote or inhibit flowering under appropriate environmental conditions. For example, Arabidopsis is a quantitative LDP with environmentally controlled pathways of photoperiod and vernalization; yet, it is also considered facultative because it will flower in the absence of environmental input under autonomous developmental control, even in complete darkness. This response suggests that under appropriate conditions, the flower-inducing substance can be made in complete darkness, as previously reported (Leopold, 1949). In Arabidopsis, the genetic control of flowering time is very complex, but great progress has been made using map-based cloning, mutant analysis, and reverse genetic approaches to identify and characterize the key regulatory genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) (Putterill et al., 1995; Fowler et al., 1999; Park et al., 1999; Kardailsky et al., 1999; Kobayashi et al., 1999). The prevailing model of photoperiodic flowering response in Arabidopsis is that light acting through phytochrome activates CO via the photoperiod-dependent pathway. CO transcription is also regulated by a circadian rhythm (Súarez-López et al., 2001). The expression and protein stability of CO in LDs but not in SDs controls the expression of FT that encodes the mobile floral signal required for floral induction (An et al., 2004; Corbesier et al., 2007). Thus in Arabidopsis, CO protein is a key regulator of flowering, and FT expression is dependent on exposure of plants to light (LD) and on the circadian rhythm of CO expression. Furthermore, FT transcription itself shows a circadian rhythm under continuous light and is thus a clock-controlled gene (Valverde et al., 2004).
The study of rice, a facultative, quantitative SDP, has increased our understanding of flowering in SDPs and LDPs. The major genes GI, CO, and FT that act in the photoperiod pathway in Arabidopsis are highly conserved in rice; however, their specific regulation has been altered by evolution to promote flowering under short-day conditions. For example, the CO ortholog in rice, Hd1, also regulates transcription of the FT ortholog, Hd3a, leading to photoperiodic flowering under SDs (Yano et al., 2000; Yano et al., 2001; Izawa et al., 2003; Kojima et al., 2002). Furthermore, it is known in rice that phytochrome B (phyB) is the main phytochrome that controls flowering (Izawa et al., 2000). PhyB acts by repressing the expression of the FT ortholog, Hd3a (Izawa et al., 2002), which is of particular interest to this study of phytochromes in another SDP. The role of phyB has been demonstrated conclusively by examining the effect of one or more brief irradiations with light during the inductive dark night, referred to as the night break (NB) effect, using a phyB mutant in which the NB inhibition of flowering and suppression of Hd3a transcription were abolished by a mutation that affected only phyB (Ishikawa et al., 2005). Other work on rice phytochromes suggests a role for both phyB and phyC, but not phyA, under long-day photoperiods; thus phyA has little effect on flowering in rice (Takano et al., 2005), in contrast to the situation in Arabidopsis (Franklin et al., 2007).
In terms of physiological systems, the best-studied SDP and the experimental subject of this investigation is Pharbitis nil Choisy strain Violet (Convolvulaceae), the Japanese morning glory (Imamura, 1967; Takimoto, 1967; Vince-Prue and Gressel, 1985). Pharbitis nil strain Violet is an obligate, qualitative photoperiodic SDP that has been used historically in research for about 80 years. In this SDP, a single inductive long night applied to photoresponsive tissue (cotyledons in 4-d to 5 d-old seedlings) initiates the phytochrome-mediated processes of floral induction. A brief irradiation during a light-sensitive phase of the inductive dark period inhibits photoperiodic flower induction in this SDP by preventing attainment of the CNL. One or more brief irradiations with light during the inductive dark night serves as an effective NB treatment (Hamner and Bonner, 1938). The NB has been used as an experimental manipulation to suppress photoperiodic floral induction and is the "litmus test" for whether an event is associated specifically with floral induction. The NB is most effective when wavelengths of red (R) light are used, and its inhibitory effect can be overturned by exposure to far-red (FR) light if applied immediately and in sufficient quanta after R light treatment (Borthwick et al., 1952; Takimoto and Hamner, 1965). The photoreversibility by FR light of the NB effect demonstrates unequivocally that the NB phenomenon is mediated though the phytochrome system. The NB effect has been used in both physiological and molecular research to unravel the complicated process of photoperiodic timekeeping that enables plants to know when to flower seasonally.
Comparison of the photoperiodic mechanisms of Pharbitis, Arabidopsis, and rice has led to an important conclusion, specifically, that Pharbitis presents a novel and different mechanism from that reported for the described model genetic systems (Eckardt, 2007; Hayama et al., 2007). The photoperiodic mechanism governing floral induction in Pharbitis is different in several important respects. First, the Pharbitis orthologs of Arabidopsis CO, Pn FT1 and Pn FT2, exhibit robust cycling of gene expression under constant darkness but do not exhibit strong expression (or circadian regulation) under continuous light. Second, the light-to-dark transition activates Pn FT expression and sets the phase of the circadian rhythm of its expression, similar to that reported here for several PHY genes. NB treatment that suppresses flowering also inhibits FT expression, as reported for rice (Ishikawa et al., 2005). Furthermore, the pattern of expression of Pn CO, the closest homolog of At CO identified thus far, was not highly correlated with the expression of the two Pn FTs under darkness because peak expression levels did not coincide except under SD treatment, leading to the conclusion that the CO ortholog, Pn CO, is not the principal regulator of FT expression in Pharbitis, and thus Pharbitis has a different mechanism than Arabidopsis and rice.
The function of CO in Pharbitis is not known. Because CO regulates FT in other photoperiodic species, the possibility remains that some unidentified Pharbitis CO gene does indeed regulate Pn FT. Also, Liu et al. (2001) reported that their Pharbitis CO ortholog could complement an Arabidopsis late-flowering co mutant. Thus, direct testing using overexpression studies with other candidate CO genes will be necessary to determine if another Pharbitis CO ortholog regulates the Pn FTs (Hayama et al., 2007). In conclusion, it appears that Pn CO and Pn FT expression are not correlated in Pharbitis as they are in the LDP model Arabidopsis and in the SDP model rice, so that "Pharbitis takes a different approach" in measuring daylength (Eckardt, 2007, p. 2968). On the basis of these intriguing findings, the use of Pharbitis as a system for molecular investigations will continue to provide new insights into the photoperiodic mechanism of flowering.
In Pharbitis also, the nature of the timekeeping mechanisms underlying photoperiodic flowering have been an intense focus of research involving the study of phytochrome. Two mechanisms have been proposed: (1) an hourglass timing mechanism based on dark reversion of phytochrome in the FR light-absorbing form (Pfr) to phytochrome in the red-light-absorbing form (Pr) (Borthwick et al., 1952; Hendricks, 1960) and (2) an endogenous rhythm mechanism that interacts with light (Bünning, 1936; Hamner, 1958; Hamner and Takimoto, 1964; Paraska and Spector, 1974; Takimoto and Saji, 1984; Vince-Prue, 1986, 1989; Lumsden, 1991). The hourglass mechanism of photoperiodic timekeeping, as originally formulated (Hendricks, 1960), was largely discounted because of the lack of a correlation between the simple decay of bulk Pfr in the dark with attainment of the critical night length (CNL) and because of the inability to account for observed interactions among light, the photoperiodic response, and the underlying endogenous rhythms. Experiments using Pharbitis conclusively demonstrated that a simple decay of bulk Pfr levels could not be correlated with photoperiodic time keeping (King et al., 1978). In addition, the instantaneous reduction of Pfr to near-zero levels by an end-of-day application of FR light did not alter the CNL in this SDP, as would be predicted if the only timekeeping mechanism relied on the dark reversion of Pfr to Pr (Vince-Prue, 1975, 1986). These findings suggest that another short-day factor must be involved as well, as proposed in the hypothetical model shown later in Fig. 11.
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; Hamner, 1958; Hamner and Takimoto, 1964; Lumsden et al., 1982; Takimoto and Saji, 1984; Vince-Prue, 1986). The complicated nature of the photoperiodic timing mechanism is illustrated by the photoperiodic responses of Pharbitis, in which the timing of the CNL and NB response is controlled by a rhythm that is initiated or reset at the onset of the main light period. In the light, the rhythm is suspended until the onset of darkness when the rhythm is released from suspension to run free (Lumsden et al., 1982; Vince-Prue, 1986, 1989; Lumsden, 1991). Others have proposed that Pharbitis has two separate rhythms involved in photoperiodic time keeping: (1) a "light-off rhythm" that controls both the time and maximum effectiveness of the NB, and (2) a "light-on rhythm" that controls the timing of the CNL and influences NB responses (Takimoto and Saji, 1984). In both cases, the rhythms approximate a 24-h cycle and thus are considered to be circadian rhythms (Bünning, 1936). This well-documented phenomenon of rhythmic photosensitivity has led to the idea that photoperiodic flower induction is a case of a circadian rhythm that interacts with light via phytochrome-controlled processes for which a model was proposed (Vince-Prue, 1989; Lumsden, 1991).
According to this model, a major pool of phytochrome establishes the phase of the circadian rhythm that underlies photoperiodic time measurement by releasing an endogenous oscillator to run free upon the transition to darkness. Light interacts with the underlying rhythm during the light-sensitive phase to inhibit flowering, which accounts for the rhythmicity of sensitivity to the NB. Light can also shift the phase of the free-running rhythm by either advancing or delaying it, dependent on the timing of the NB. To fully account for the interaction of phytochrome and the endogenous circadian rhythm, the model also proposes the presence of two pools of phytochrome with separate actions during photoperiodic induction: a stable pool of Pfr required for induction and an unstable pool of Pfr involved in the NB and initial perception of dark (Vince-Prue, 1989; Lumsden, 1991; Thomas, 1991). The identification of multiple phytochrome genes in many plant species provides a molecular explanation for the proposed action of functionally distinct phytochrome pools.
In our earlier work on the molecular basis of flowering in Pharbitis, we identified mRNAs whose patterns of abundance are under both light and clock control and correlate with the photoperiodic induction of flowering (Zheng et al., 1993, 1998; O’Neill et al., 1994). The recent interest in using Pharbitis for molecular genetic studies of its orthologs of Arabidopsis flowering time genes illustrates the importance of this SDP as a model system for photoperiodic flowering (Liu et al., 2001; Kim et al., 2003; Hayama et al., 2007). Dark activation of clock control, dark induction of transcription, and circadian regulation were reported for each of these regulatory genes. These findings are consistent with earlier physiological findings in Pharbitis that the rhythm of expression of photoperiodic responses is set at the time of transfer to darkness (subjective dusk). Thus, the biological clock that regulates expression of CO and FT is entrained by the light-to-dark transition that occurs at dusk. Hayama et al. (2007) concluded that Pharbitis contains a light-sensitive clock that measures night length and regulates FT expression. This mechanism is compatible with that identified here for the PHY genes, and possibly for their corresponding proteins that we have not examined yet, and thus has important possibilities for increasing our understanding of the underlying mechanism of photoperiodic timekeeping.
The regulation, expression, and action of PHY genes and their proteins are of fundamental interest to the field of flowering. Molecularly, phytochromes comprise a family of nuclear-encoded photoreceptors (Quail, 1991, 2002; Furuya, 1993; Sullivan and Deng, 2003; Franklin et al., 2005). In angiosperms, five classes of phytochromes, designated phytochrome A through E (phyA–phyE), have been identified. The identification of multiple phytochrome genes in higher plants suggests a potential molecular mechanism for the unexplained multiple phytochrome pools inferred from physiological experiments of the photoperiodic control of flowering in SDPs. For example, the early identification of PHYA and PHYB genes in both eudicots and monocots and the determination of their special functions in photomorphogenesis led early on to the suggestion that their gene products contribute to two physiologically distinct pools of phytochrome (Tokuhisa et al., 1985; Christensen and Quail, 1989; Kay et al., 1989; Heyer and Gatz, 1992; Dehesh et al., 1993;). This idea has been extended even further by the discovery that many species have more than two phytochrome genes, as originally discovered in Arabidopsis and Lycopersicon (Sharrock and Quail, 1989; Clack et al., 1994; Hauser et al., 1995). To date, PHYA, PHYB, and PHYC genes have been identified in both eudicots and monocots, while PHYE is known only from eudicots and PHYD only from Arabidopsis. Phytochrome orthologs have also been identified from gymnosperms, seedless vascular plants, bryophytes, and green algae, in some cases, with multiple distinct forms present in a single species. Based on the existence of multiple, discrete molecular species of phytochrome and marked diversity among their amino acid sequences in Arabidopsis and rice, it is now known that each phytochrome can play a distinct role in light-mediated plant development (Smith and Whitelam, 1990; Quail, 1991; Furuya, 1993; Izawa et al, 2000; Quail, 2002; Izawa et al, 2003; Sullivan and Deng, 2003; Ausín et al., 2005; Franklin et al, 2005; Takano et al., 2005; Thomas, 2006; Franklin et al, 2007).
Specific photomorphogenic responses have been assigned to specific phytochromes, with most of these assignments based on studies of Arabidopsis and rice. Thus far, only a few studies have reported molecular-level information about Pharbitis phytochrome. Szmidt-Jaworska et al. (2000) used antibodies to study phyA levels in P. nil and concluded that phyA does not directly control photoperiodic floral induction. Carter et al. (2000) examined phytochrome regulation of PHYA mRNA levels in P. nil, and based on the known responses of this SDP to various R and FR light treatments that inhibit flowering and their dramatically different effects on PHYA transcript accumulation, the authors concluded that there was no evidence to support the hypothesis that PHYA mRNA levels play a direct role in the flowering response of Pharbitis. Some other factors, such as the control of nuclear import of phyA or a "phytochrome-interacting factor" might be important in regulating phyA action and its putative role in the control of flowering in Pharbitis.
The research presented here builds on our earlier molecular research on Pharbitis and has the overall goal of examining whether the expression of four different phytochrome genes could explain some of the photoperiodic flowering response in this SDP, building upon and adding to the body of knowledge for this classic photoperiodic species. To assess the potential role of multiple phytochrome species in photoperiodic timekeeping, we characterized some of the molecular events at the level of mRNAs of the different phytochrome genes that could possibly control photoperiodic flower induction. We wish to emphasize that we did not investigate the corresponding phytochrome proteins in parallel, although this would be a worthy pursuit. Here we report the identification and characterization of four distinct Pharbitis phytochrome cDNAs, designated PHYA, PHYB, PHYC, and PHYE, representing the four different PHY genes of the Pharbitis phytochrome family. One or more of the encoded proteins of these distinct PHY genes may contribute to the physiologically distinct phytochrome pools previously described for this species. In addition, the circadian regulation of certain of these PHY genes during dark induction may provide part of the molecular basis for the linkage between day length and an endogenous clock in the photoperiodic control of flowering. We hope the current report will provide a valuable complement to other research that utilizes P. nil strain Violet for molecular studies of the photoperiodic control of flowering. Finally, we propose a model that integrates the known physiological with the molecular events that occur during the photoperiodic control of flowering in SDPs.
MATERIALS AND METHODS
Plant material
Pharbitis nil Choisy strain Violet (Japanese morning glory, also known as Ipomoea nil; Convolvulaceae) was used for all experiments. According to the International Code of Botanical Nomenclature, the species is correctly named Ipomoea nil, due to a change in the generic designation from Pharbitis to Ipomoea; however, in this report, we have chosen to use the original generic name Pharbitis in keeping with the large body of historical and current literature on this SDP. Seeds of this Pharbitis strain were originally obtained from Marutane Co. (Kyoto, Japan) over 30 years ago and have been maintained as a selfing population in the greenhouses of the University of California, Davis. The seed population used for all research reported here was derived from a large founding population of plants that had a strong terminal flowering response to a single inductive night treatment. Terminal flowering lines were selected and inbred for over 20 generations. This population of seeds is less genetically heterozygous than seeds obtained directly from Marutane Co., which supplies seeds from field-grown, open-pollinated plants that have a variable terminal flowering response. Seedling plants derived from our genetically homogeneous UC Davis line show complete terminal flowering after 14 h of darkness and thus have a strong qualitative and uniform response needed for the molecular work.
Seedling preparation, growth, and photoperiodic treatments
Seedling preparation, growth, and photoperiodic treatments were as described previously by O’Neill et al. (1994). In brief, seeds were scarified in concentrated sulfuric acid for 45 min on a stir plate, rinsed in deionized water and imbibed in aerated distilled water overnight. The germinated seeds were planted in trays containing a standard soil mixture and held in a growth chamber under continuous fluorescent light (250 µmol•m–2•s–1; Phillips VHO/EW 185 W/1500 mA) at 27 ± 2°C. Seedlings were watered twice daily with 0.5x Hoagland’s nutrient solution. Any late-germinating seedlings were removed from the trays to obtain a uniformly developed population of seedlings for experimental treatment. Photoperiodic treatments were routinely initiated at 1630 hours (0 h) at 6 d post germination.
Six-day-old seedlings grown in continuous white light were subjected to the indicated light treatments. Cotyledon blade-only tissue, roots, and hypocotyls were harvested in complete darkness directly into liquid nitrogen, transferred to lightproof plastic containers, and stored at –80°C for later extraction of RNA. For the dark time-course experiments, cotyledon tissue (also minus any plumule or hypocotyl tissue) was harvested during dark treatment at 4-h intervals from 0 to 48 h in complete darkness directly into liquid nitrogen, transferred to lightproof plastic containers, and stored at –80°C for later extraction of RNA. For each experimental time point, a minimum of 10 plants were returned to the growth chamber for an additional 2–3 weeks of growth in continuous light. After 10 d, the effectiveness of each treatment in inducing flowering was determined by examining each node. Flowering was assessed by determining the number of flower buds per plant, the percentage of flower buds/total buds, the percentage of plants possessing flower buds, and the percentage of plants with terminal flower buds.
Photoperiodic and light treatments
Photoperiodic light treatments were initiated at 1630 hours (0 h) on 6-d-old seedlings. Treatments included (1) an extended dark treatment of up to 48 h, (2) a night break (NB) with 10 min of R light interruption given at 8 h into the dark period, and (3) an end-of-day treatment with 10 min each of R, FR, R/FR, or FR/R, prior to the transfer to darkness. Red light treatments were provided by exposure to four fluorescent tubes (Philips F30T12/CW/RS) filtered with a 3-mm-thick red plexiglass sheet (Acrylite GP, color 210–0; CYRO Industries, Mount Arlington, New Jersey, USA) as previously described (O’Neill et al., 1994). The FR light source used for actinic irradiation was provided by two fluorescent tubes (Sylvania F48T12/660 nm/VHO) filtered with a 3-mm-thick far-red plexiglass sheet (FRS700 plexiglas, Rohm and Haas dye no. 58015, Hayward, California, USA) as described by Li and Lagarias (1994). At the end of each treatment, seedling tissues were harvested in complete darkness, frozen in liquid nitrogen, and transferred to –80°C for storage.
Genomic DNA and RNA isolation
Genomic DNA was isolated from 20 g of frozen pulverized cotyledon tissue according to the procedure of Jofuku and Goldberg (1988). Total RNA was isolated from separately harvested organs of Pharbitis nil seedlings as described previously (O’Neill et al., 1994). Poly(A)+ RNA was subsequently isolated by oligo(dT)- cellulose chromatography (Pfizer, New York, New York, USA). Typical total RNA yields from cotyledon tissue was 100 µg/g fresh mass of tissue, with a poly(A)+ RNA yield of approximately 1.5% of total RNA.
Polymerase chain reaction (PCR) amplification
PCR experiments were performed using both Pharbitis nil genomic DNA and cotyledon cDNA as the template. RNA used for RT-PCR experiments was isolated from 12-h dark-induced cotyledon tissue. The degenerate upstream (5'-ATGGKNTAYAARTTYCAYGA-3') and downstream (5'-ARRAAYTCRCANGCRTANCK) PCR primers were designed to hybridize to two conserved regions flanking the chromophore attachment site of the phytochrome apoprotein (Sharrock and Quail, 1989). Standard reaction conditions were as follows: 40 cycles of 1 min denaturing at 94°C, 2 min annealing at 37°C, and 1 min elongation at 72°C. The primary PCR products were separated by electrophoresis in 0.7% low-melt agarose. The PCR products were purified and cloned into the SmaI site of pBluescript KS+ II (Stratagene, La Jolla, California, USA) or into the TA PCR cloning vector (Invitrogen, Carlsbad, California, USA).
cDNA library construction
Total RNA was isolated as described previously (O’Neill et al., 1994). Poly(A)+ RNA was isolated using paramagnetic oligo(dT) beads (Dynabeads, Dynal/Invitrogen, Oslo, Norway) according to the manufacturer’s suggestions. LiCl was removed from the poly(A)+ RNA by two ethanol precipitations prior to first-strand cDNA synthesis. Libraries were constructed from 5 µg poly(A)+ RNA isolated from 6-d-old cotyledon tissue treated with 8–12 h of darkness. The cDNA was constructed and cloned into the 
ZAPII phage vector (Stratagene) according to the manufacturer’s procedures. The coyledon-specific cDNA library contained 
3 x 106 clones, 95% of which contained long inserts.
cDNA library screening
The cotyledon-specific cDNA library was screened by plaque hybridization as follows: for primary screening, 100000 plaques were screened using 32P-labeled PCR products of PHYA, PHYB, PHYC, and PHYE as probes as described previously (O’Neill et al., 1994). In brief, filters were prehybridized at 40°C in prehybridization solution (50% deionized formamide, 2x PIPES, 0.5% SDS and 100 µg denatured sonicated salmon sperm sssDNA/ml) for 10 h and hybridized for 40 h in hybridization solution (50% deionized formamide, 2x PIPES, 0.5% SDS, 100 µg denatured sssDNA/ml and 106 cpm probes/ml). Hybridized filters were washed twice at 45°C in 1x SSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS solution for 30 min. each, and then exposed to Kodak (Rochester, New York, USA) XAR-5 x-ray film with one intensifying screen (Cronex Lightning Plus, DuPont, Wilmington, Delaware, USA) overnight at –80°C. Individual positive plaques were purified by secondary and tertiary screening or rounds of plating and hybridization. The phagemid pBluescript KS+ containing the cDNA insert was isolated from the phage clone by in vivo excision for further analysis. Sets of cDNA clones containing each of the PHY transcripts were obtained and stored for further analysis.
DNA sequence analysis
DNA sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977) using a Sequenase Sequencing System (U.S. Biochemical Corp., Cleveland, Ohio, USA). The large fragments were sequenced by constructing a nested set of deletion plasmids using the Erase-a-Base system (Promega, Madison, Wisconsin, USA). The nucleotide sequences were determined from overlapping clones; 100% of the sequence was determined from both strands. Nucleotide and predicted amino acid sequence analysis and database homology searching were performed using the Genetics Computer Group program (University of Wisconsin, Madison, WI) or NCBI programs.
Phylogenetic analysis
Seventy-six phytochrome and phytochrome-like sequences from green algae and land plants (refer to Appendix 1), identified using BLAST (Altschul et al., 1990) searches, were downloaded and aligned using the program Clustal_X (Thompson et al., 1997). Phylogenetic analyses of the aligned amino acid sequences based on maximum parsimony were implemented in the program PAUP* (Swofford, 2002) with heuristic searches using the tree-bisection-reconnection (TBR) branch-swapping algorithm and 1000 random taxon addition replicates and no limit on the number of trees saved. The resulting trees were rooted with the green algal sequences, and a strict consensus tree was constructed. Relative support for clades was assessed using 1000 bootstrap replicates with 10 random taxon addition replicates per bootstrap replicate and the number of trees saved per replicate capped at 100.
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). The membrane was prehybridized and hybridized with radiolabeled probe at 42°C in the buffer solution described by Zheng et al. (1993) and washed two times in 1x SSC, 0.5% SDS at 50°C. The same DNA blot was used sequentially for hybridization with PHYA, PHYB, PHYC, and PHYE cDNA probes.
Total RNA was fractionated by electrophoresis in formaldehyde agarose gels and transferred to a nylon membrane. RNA gel blots were probed with 32P-labeled DNA fragments at normalized amounts labeled to high specific activity. Prehybridization, hybridization, and washing of blots was carried out as previously described (O’Neill et al., 1994). Autoradiography was carried out at –80°C using preflashed Kodak XAR-5 film and a single intensifying screen. The hybridization signal was quantified from autoradiographs using the Bio Image (Millipore Corp., Billerica, Massachusetts, USA) analytical imaging instrument equipped with a video camera, digital image processing, and data processing. The software used was Visage Release 4.6K on a SPARC station computer system (UNIX). For the extended-dark time course, the relative hybridization signal was expressed relative to the maximum signal on each individual autoradiograph and against control signal. The autoradiographic exposure times for RNA blots were approximately equivalent for PHYB, PHYC, and PHYE. Depending on the experimental treatment affecting the abundance of PHYA mRNA, the autoradiographic exposure times had to be modified to reflect a semiquantitative or relative difference in transcript levels among the four PHY genes.
RESULTS
Pharbitis nil: A model photoperiodic system
To explore the hypothesis that the multiple phytochrome pools proposed to contribute to photoperiodic regulation of flowering corresponded to the products of distinct phytochrome genes, we chose to study the phytochrome genes of a model photoperiodic system, Pharbitis nil Choisy strain Violet (Japanese morning glory). This particular strain of Pharbitis is an obligate, qualitative SDP that has been extensively characterized with regard to the physiology and biochemistry of photoperiodic floral induction (Imamura, 1967; Takimoto, 1967; Saji et al., 1983; Vince-Prue and Gressel, 1985). Seedlings become fully responsive to photoperiodic treatment within 5 d of germination, as indicated by a terminal flowering response (O’Neill et al., 1994). In P. nil strain Violet seedlings, flowering is fully induced by exposure of the cotyledons to a period of darkness that exceeds a critical length, also called the CNL (Imamura and Takimoto, 1955). Figure 2A shows that in a genetically homogeneous population of these seedlings, flower induction is stimulated by a single dark period that exceeds 11 h with the response being completely saturated by 14 h. In addition, the inductive effects of dark treatment can be fully reversed by a brief exposure to red light, the night break (NB) treatment, given at exactly 8 h into the dark period (O’Neill et al., 1994), which provides a useful negative control for experimental treatments that induce flowering, as shown in Fig. 2B. The precise control of flowering by a single critical dark period, its inhibition by NB, and the competency to flower at an early developmental stage make this SDP an ideal subject for examining the molecular basis of phytochrome involvement in the photoperiodic control of flowering.
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; Kay et al., 1989; Thummler et al., 1992). The deduced amino acid and nucleotide sequence identities among the four PCR products range from 65 to 75%. The deduced amino acid sequences revealed the presence of a notable divergent region close to the chromophore attachment site in both the P. nil and Arabidopsis phytochromes (Fig. 3A, B).
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), the open reading frame likely starts at nucleotide 90, although there is another in-frame translation initiation codon AUG at nucleotide 43, suggesting the possibility of multiple transcription start sites. Figure 4 illustrates that the predicted polypeptide of 1115 amino acids is closely related to Arabidopsis phyE, although there are three divergent regions between P. nil and Arabidopsis phyE amino acid sequences (boxed regions). As with Arabidopsis phyB, C, D, and E, the deduced polypeptide of P. nil phyE does not contain a PEST motif that would lead to rapid protein turnover (Rogers et al., 1986; Chevaillier, 1993), suggesting that the Pfr form of Pharbitis phyE may be relatively stable.
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; Heyer and Gatz, 1992; Dehesh et al., 1993; Clack et al., 1994). In addition, the PHY gene fragment probes did not cross-hybridize with each other, indicating that each probe could be used to specifically assess the mRNA abundance of each transcript. Analysis of transcripts sizes showed that the various PHY genes encode mRNAs of different final sizes ranging from 4.2 to 4.6 kb, as shown in Fig. 7.
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Phytochrome mRNAs are differentially regulated by light in Pharbitis nil cotyledons
Previous studies have shown that PHYA mRNA abundance is downregulated by light and that its gene product corresponds to type I (light labile) phytochrome (Sharrock and Quail, 1989). The other PHY genes and their gene products are thought to be more stable in light and thus are considered members of the type II (light stable) group (Quail, 1991, Quail, 2002; Furuya, 1993). To determine the effect of light on the expression of the four P. nil PHY genes and to compare the relative abundance of their transcripts under specific light treatments that affect flowering, we analyzed the levels of mRNA in seedling tissue following dark and various light treatments. Figure 8 shows that PHYA mRNA is the most abundant phytochrome transcript in etiolated cotyledons of dark-grown seedlings compared to other PHY genes and the control probes (PNCAB and PNACTIN). Moreover, the level of PHYA mRNA was reduced by a 10-min R light treatment. A FR light treatment immediately after a R light treatment reversed the inhibitory effect of the R light, thereby establishing the photoreversibility of this response at the level of transcription. Far-red light treatment also caused a decrease in PHYA. For PHYA, the R/FR treatment may not have been complicated by the effect of FR after R treatment, given that both have a depressive effect on PHYA mRNA levels. Finally, PHYA transcription dramatically decreased in light-grown tissue sometime after 10 min of white light. The lower level of transcript after 1 min of white light as opposed to the 10-min time point is possibly an experimental artifact or due to some immediate reaction to transfer to white light that is temporarily compensated for within the first 10 min prior to adjustment by prolonged exposure to light. The level of signal of the actin control would support the latter explanation. PHYA transcript was barely detectable in continuous white light (CL), as compared to all other PHY genes; however, of the four PHY genes, its abundance, when in continuous darkness, reached the highest levels in cotyledons. These responses are consistent with PHYA encoding a type I (light labile) phytochrome.
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To confirm that each lane contained an equivalent amount of RNA, we probed the RNA blot with a P. nil ACTIN gene that has been reported previously to be insensitive to light, as well as with a P. nil CAB gene, whose mRNA levels were reported to be upregulated by light and regulated by an endogenous clock (also see Fig. 10; for additional detailed expression patterns, see O’Neill et al., 1994). Overall, the differences in PHY gene expression are striking and provide support that the patterns of PHY expression reflect true differences in phytochrome gene expression. From these results, it can be concluded that in P. nil, there are at least two pools of phytochrome mRNA with different patterns of abundance in light and darkness: one pool is light labile and responsive to R and FR light (PHYA), whereas the other is not light labile and is not noticeably responsive to white, R, or FR light (PHYB, PHYC, and PHYE). In addition, PHYC mRNA may be intermediate in its stability in light.
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). Figure 9 illustrates the effect of a 10-min R light–NB treatment, applied precisely at 8 h into an inductive dark period (subjective night), on the abundance of each of the four PHY mRNAs at time intervals subsequent to the NB at 12, 16, and 20 h in dark-induced and NB-treated cotyledon tissue. As with floral induction, NB treatment also strongly inhibited PHYA, PHYB, and PHYE mRNA accumulation in cotyledons. By comparison, PHYC was less sensitive to NB at all time points examined. For PHYA, the effects of dark and NB treatment were similar at each of the three time points sampled, as well as at later time points (data not shown). However, the levels of PHYB and PHYE mRNA declined during extended darkness (by 20 h), with NB having less effect on altering mRNA levels at the later time point. The latter result is suggestive that the transcription of these two PHY genes, but not PHYA, is regulated by an endogenous clock during extended darkness that coincides with subjective dawn. As a positive control, the RNA gel blot was probed with the HIGH MOBILITY GROUP1 (HMG1) gene that encodes a transcriptional regulator whose abundance is enhanced by NB treatment over even the dark-induced level (Zheng et al., 1993; O’Neill and Zheng, 1998), as was also observed in this experiment.
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24 h. PHYE expression peaked at 4, 32, and 56 h of darkness, with lowest levels of expression at 16, 48, and after 60 h. CAB gene transcription is known to be light-regulated through the phytochrome system and by an endogenous clock that exerts circadian control (Silverthorne and Tobin, 1984). For the P. nil CAB gene, this type of expression pattern has also been reported (O’Neill et al., 1994). Subjective dawn would be around 12 h into the dark treatment, at which time CAB gene expression increased (Fig. 10). Peak levels of CAB expression were observed at 16 and 40 h of darkness, with dampened levels of expression observed after that time. PHYE and CAB gene expression show opposite patterns of peaks and troughs, with PHYE increasing when CAB gene expression is declining. A similar pattern of rhythmic expression was observed for PHYB and PHYC (data not shown). We could not detect any significant expression of PHYA in these experiments on light-grown seedlings after a number of independent replications. Thus, in P. nil cotyledons, the free-running pattern of expression of several phytochrome genes except PHYA in continuous dark is regulated by an endogenous clock that imposes a circadian rhythm on gene expression. DISCUSSION
Physiology of photoperiodic flowering and phytochrome genes
The earliest model of phytochrome action in SDP was that Pfr levels, established in the light, decayed in the dark to a critical low level that triggered the flowering response (Borthwick et al., 1952; Hendricks, 1960). This model of photoperiodic timekeeping was challenged by spectrophotometric assays of the phytochrome in P. nil that demonstrated that the major pool of Pfr decayed within 2 h after the onset of dark, but flowering was not induced until 10 h later (King et al., 1978). In addition to this major unstable pool of spectrophotometrically detected Pfr, there is physiological evidence for a more stable Pfr pool that participates in flower induction in SDPs. The stable Pfr was inferred from end-of-day exposure to FR light, a treatment that inhibits flowering in many SDPs. These results have collectively been interpreted to indicate the presence in SDPs of at least two differentially stable pools of phytochrome that contribute to photoperiodic timekeeping (Vince-Prue, 1989). The question still remains as to how phytochrome participates in photoperiodic timekeeping.
Phytochromes have both distinct and overlapping roles in photomorphogenesis
The photoregulatory roles of different phytochromes in plant growth and development has been addressed in phytochrome mutants and in transgenic plants, mainly using Arabidopsis. Each phytochrome plays a distinct major role in photomorphogeneis, and some phytochromes have overlapping functions (Franklin et al., 2003, 2005, 2007; Sullivan and Deng, 2003; Takano et al., 2005; Thomas, 2006). Because this study is concerned with the role of phytochromes in photoperiodic flowering, the present state of our knowledge about each known phytochrome is briefly discussed.
Phytochrome A (phyA) is considered the light-labile or type I phytochrome (Quail, 1991, Quail, 2002; Furuya, 1993) involved in seed germination, hypocotyl growth inhibition, and de-etiolation during early photomorphogenesis. Phytochrome A accumulates as Pfr to relatively high levels in dark-grown, etiolated tissues, but unlike other phytochromes its levels are rapidly depleted upon exposure to white or R light due to light-induced proteolytic degradation of Pfr and to downregulation of PHYA gene expression (Quail, 1991, 2002). The PHYA gene can be structurally complex with three potential transcriptional start sites and different gene products (Quail, 2002). Early analysis of Arabidopsis mutants affecting phytochrome function revealed that phyA is responsible for sensing a FR-enriched light environment (Johnson et al., 1994; Parks and Quail, 1993; Nagatani et al., 1993; Whitelam et al., 1993). While the primary function of phyA is de-etiolation in continuous FR light, it has some overlapping functions with phyB in the shade avoidance response (Cantón and Quail, 1999). In addition, the analysis of photoresponses of light-grown phyA mutants of Arabidopsis has demonstrated that phyA plays a key role in the perception of daylength (Bagnall et al., 1995). Most recently, Franklin et al. (2007) have shown that phyA has activity at higher R photon irradiances than previously known and thus can function as an effective irradiance sensor that may contribute to the regulation of growth and development in daylight-grown plants. Collectively, these studies suggest that in Arabidopsis, phyA is the most important phytochrome species for photoperiodic flowering.
In contrast, phytochrome B (phyB) is a light-stable or type II photoreceptor that senses a R-enriched light environment (Quail, 1991, Quail, 2002; Furuya, 1993). Work on phyB mutants of Arabidopsis has demonstrated that phyB acts in the shade avoidance pathway and not in the photoperiodic pathway (Goto et al., 1991; Nagatani et al., 1991; Devlin et al., 1992; McCormac et al., 1992; Reed et al., 1993; Halliday et al., 1994; Bagnall et al., 1995). But also in Arabidopsis, phyB regulates the abundance of CONSTANS (CO) protein posttranslationally, with low levels of CO functioning to inhibit photoperiodic flowering (Valverde et al., 2004). In the phyB mutant, CO is maintained at a high level, which leads to the promotion of the flowering regulator FLOWERING LOCUS T (FT) expression and FT signaling to induce flowering (Samach et al., 2000). Thus in Arabidopsis phyB has a repressive effect on floral induction. However, phyB is the major phytochrome that senses daylength in the short-day plants rice, sorghum, and potato (Heyer and Gatz, 1992; Childs et al., 1997; Izawa et al., 2000; Takano et al., 2005).
Characterization of phyC null mutants of Arabidopsis has shown that phytochrome C (phyC) has multiple, complex functions. PhyC is involved in photomorphogenesis throughout the plant’s life cycle, with a photosensory specificity similar to that of the light-stable phyB, phyD, and phyE, but with a more complex pattern of differential crosstalk with phyA and phyB (Franklin et al., 2003; Monte et al., 2003). Recently, Balasubramanian et al. (2006) reported that allelic variation at the PHYC locus affects flowering-time traits in natural populations of A. thaliana, linking this gene with latitudinal variations in flowering time.
PHYD was identified in Arabidopsis as one of the five main phytochrome species (Sharrock and Quail, 1989). Using Arabidopsis phyD mutants, Devlin et al. (1999) determined that phyD acts in the shade avoidance pathway by controlling flowering time and leaf area and further that phyC and/or phyE also act in concert with phyD. In Arabidopsis, the phyD and phyB proteins share 80% amino acid identity and are believed to have arisen from a recent gene duplication event in the Cruciferae. Further, phyD and phyB are more closely related to phyE (55% identity) than to phyA or phyC (47% identity) (Mathews and Sharrock, 1997).
The PHYE gene was also originally identified in Arabidopsis (Sharrock and Quail, 1989; Clack et al., 1994). Based on double mutant analysis, phytochrome E (phyE) acts in conjunction with phyB, and to a lesser extent with phyD, in the shade avoidance pathway (Devlin et al., 1998). PhyE also has a role in seed germination along with phyB but not with phyD, something that identifies a novel function for phyE relative to other phytochromes (Hennig et al., 2002). Halliday and Whitelam (2003) have also shown that cooler temperature and photoperiod can alter the relative contributions of phytochromes, including phyE relative to phyB, to flowering, thus these genes are also candidates for those controlling photoperiodic flowering.
Pharbitis nil phytochrome genes comprise a small divergent family
Here we present evidence that the model SDP, Pharbitis nil, contains at least four distinct phytochrome genes that are predicted by sequence analysis to encode distinct molecular entities and by expression analyses to have different photobiological responses. These results are consistent with the physiological and biochemical evidence for multiple phytochrome pools in P. nil. Thus, we suggest that these divergent PHY genes represent the molecular basis of physiologically distinct phytochrome pools. Although more than two PHY genes have been cloned from only a small number of species thus far (for example, from Arabidopsis, Lycopersicon, and now Pharbitis), it appears that the presence of more than one PHY gene is a common feature of plants (full information in Appendix 1), which makes our hypothesis reasonable. The PHY genes of Pharbitis are sufficiently divergent that they do not cross-hybridize under high stringency conditions. On the basis of sequence information, the Pharbitis PHY genes clearly have structural differences in similar genomic regions that account for this divergence from one another, as well as from other species, including Arabidopsis.
Phylogenetic analysis of PHY genes in green algae and land plants
The results of our phylogenetic analysis mirror those of Donoghue and Mathews (1998), which were based on fewer sequences and analysis of nucleotides rather than amino acids. As in their study, we recovered four strongly supported clades of angiosperm phytochrome genes, phyA, phyB, phyC, and phyE, and all except the phyE clade included sequences from both monocots (primarily represented by grasses) and eudicots. In addition, phyA is sister to phyC and phyE sister to phyB, with gymnosperm sequences sister to either the phyA/phyC clade or to the phyB/phyE clade. These results suggest that a single duplication occurred in an ancestral seed plant prior to the divergence of extant gymnosperms from angiosperms and that two subsequent duplications occurred in an ancestral angiosperm prior to the divergence of monocots from eudicots. Additional duplications have occurred within some lineages resulting in additional copies in some species (e.g., Zea mays). The presence of multiple phytochromes in some species of nonseed-bearing land plants indicates that duplications have occurred in those groups as well, but relationships among the basally diverging branches in our tree are too weakly resolved, and sampling is too limited, to allow us to make strong inferences about the relative timing of those events with respect to the divergence of the major lineages of bryophytes and vascular plants.
PHY genes in Pharbitis nil are differentially regulated by growth and light
In addition to a distinct phylogenetic history, each PHY gene appears to have a unique profile of regulation by growth, photoperception, and an endogenous clock, as summarized in Table 1. The properties of the PHY genes are also distinct from other light- and clock-regulated genes (CAB, HMG1) as well as from constitutive genes (ACTIN) and other important developmental genes (LEAFY) that do not exhibit the same regulation (Table 1). The comparison among the four PHY genes is striking, suggesting that the variations in PHY expression reflect functionally important differences. In light of these results, it can be concluded that in Pharbitis, there are at least two pools of phytochrome mRNA with different patterns of abundance in light and dark-treated tissue: one pool is light labile and responsive to R/FR (PHYA), whereas the other is not light labile and is not completely responsive to R/FR light under the experimental conditions applied (PHYB, PHYC, and PHYE). Furthermore, it can be suggested that one or more of the phytochromes participates differentially in photoperiodic timekeeping, and from the collective data, this does not appear to be PHYA.
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; Kay et al., 1989; Heyer and Gatz, 1992; Dehesh et al., 1993; Furuya, 1993). Our results are consistent with these previous reports. However, in contrast to Arabidopsis PHYE, which was reported to have a similar level of expression in both light- and dark-grown tissue (Clack et al., 1994), in Pharbitis, PHYE mRNA abundance was clearly elevated in light-grown tissue. On the basis of this distinction, we suggest that the PHYE gene product may comprise a novel phytochrome pool. Confirmation of this hypothesis will require additional investigation, but it is intriguing that this particular PHY gene belongs to a group of seed plant PHY genes, the phyE clade identified in this study, that has fewer members and is thus less commonly represented among phytochromes.
PHY genes are regulated by an endogenous clock with a circadian rhythm
The regulation of photoperiodic floral induction involves the interaction of light and phytochrome with an endogenous rhythm (Vince-Prue, 1986; Vince-Prue and Lumsden, 1987; O’Neill, 1992). The interaction of phytochrome with an endogenous rhythm in photoperiodic timekeeping was revealed by NB experiments, where a brief exposure to R light during the dark inductive period inhibited flowering of SDPs. The effectiveness of NB treatments was found to be highly dependent upon the time of its application during the dark period, with a sensitivity that was rhythmic (Takimoto and Ikeda, 1960; Fredericq, 1964; Takimoto and Hamner, 1965; Lumsden and Vince-Prue, 1984; O’Neill et al., 1994). The characteristics of the rhythm in NB sensitivity clearly demonstrate that phytochrome-dependent photoperiodic timekeeping interacts with an endogenous circadian rhythm (Spector and Paraska, 1973; Paraska and Spector, 1974; Heide, 1977; Lumsden et al., 1982; Lumsden and Furuya, 1986; Lumsden et al., 1986; Lumsden, 1991; O’Neill, 1992).
Interestingly, mRNA levels for PHYA, PHYB, and PHYE, but not as much for PHYC, were reduced by NB treatment during a photoinductive dark treatment. However, the abundance of PHYE, PHYB, and PHYC mRNA in cotyledons was regulated by an endogenous circadian rhythm during a prolonged dark period. This rhythmic abundance of PHY genes could be the molecular basis for the interaction between photoperiod and a circadian rhythm in regulating flower induction. Immunochemical data are needed to confirm whether functional phytochrome protein levels also demonstrate circadian regulation in Pharbitis cotyledons. On the basis of our limited data, we suggest that one or more of the non-PHYA genes (PHYC- E) contribute(s) to the phytochrome pool that participates in photoperiodic floral induction in Pharbitis. Further study of each phytochrome protein is needed to determine if changes in the protein levels mimic the changes in the PHY mRNA levels reported here. Transgenic plants with mutant or altered expression of each PHY gene, alone and in combination, will be required to test this hypothesis.
CAB gene expression in Pharbitis nil
The examination of CAB gene expression has helped us to understand better the possibilities for PHY gene activity. As with PHY, the CAB genes are expressed in the photoreceptive tissue, in this case, the cotyledon. CAB gene expression was observed in the light before dark induction, exhibiting a circadian rhythmicity, but exhibited peak expression at subjective dawn, which was not coincident with attainment of the CNL nor dark time measurement and certainly not with the rhythmic expression of other candidate genes for those involved in photoperiodic flowering. However, NB treatment reduces the expression of CAB, so it too is regulated via phytochrome. Because the expression of CAB is not specifically correlated with the photoperiodic time measurement that leads to flowering, one possibility is that CAB gene expression is entrained to a different circadian oscillator, or output from the main clock. A similar conclusion was reached for CAB in relation to the expression of Pn FTs by Hayama et al. (2007), who have suggested that if different circadian clocks with different entrainment characteristics control the rhythms of Pn FT and Pn CAB (or the same master clock with different oscillators clocks for these two different genes), then one reasonable possibility is that circadian regulation is cell autonomous in different tissues where these genes are expressed. Hayama et al. (2007) proposed that in Pharbitis, a clock system entrained by dusk acts in the phloem to regulate Pn FT in a dark-dependent manner, whereas a second system acts in the leaf mesophyll to regulate Pn CAB expression, presumably not in a dark-dependent manner. This explanation would account for our results as well.
Integrating molecular with physiological studies of Pharbitis
In Pharbitis, the circadian clock regulates PHY and FT so that peak phases of gene expression always occurs at a constant time after dusk and are suppressed by light, as shown in the NB response. These findings are consistent with physiological experiments that suggested the existence of a dark-initiated circadian rhythm in Pharbitis (Lumsden and Furuya, 1986; Lumsden, 1991). The mechanism by which PHY and FT expression are suppressed by light (dark-initiated rhythm of expression, entrainment by dark time, and NB inhibition) is not clear. Phytochrome is clearly involved though as the primary photoreceptor involved in photoperiodic flowering, and it must be regulating its own expression in concert with an endogenous clock. Moreover, other yet unidentified light- or clock-controlled genes must also be involved in dark time measurement that ultimately informs the regulation of FT.
A model for the various physiological processes and possible genes involved in photoperiodic flowering, as reported in the literature thus far, is proposed in Fig. 11. This model incorporates the possibility of another as yet unidentified gene product that interacts with Pfr phytochrome. According to the model, light of sufficient quanta must precede darkness for the darkness to be effective, consistent with known physiological studies of SDPs. In the light phytochrome in the Pfr form, energy from light, and the precursor for a Pfr-interacting factor accumulate. The production of a phytochrome bound to the phytochrome-interacting factor begins in the light but requires darkness to accumulate because of its degradation in the light. As soon as accumulation exceeds degradation, the amount of complexed phytochrome begins to increase, approximately at the time of the CNL, and at this point, NB treatment is most effective in driving levels back to zero. Once the CNL has been reached, the altered phytochrome accumulates to a level sufficient to induce a gene involved in marking short days—a "short-day-factor"—that increases in dark and mediates the short-day effects, first reaching a threshold level (partial floral induction) and if accumulation continues, then an optimal level (full floral induction). The original concept of a "short-day factor" was proposed by others (A. Kadman-Zahavi, Volcani Center, Israel, personal communication).
This simple scenario can be overlain with genes that have been identified in Pharbitis. In this case, predicted phy proteins present in the light may constitute a photoperiodic pool and participate in formation of a modified phytochrome pool complex. CAB genes are also functioning, contributing to the accumulation of substances needed in the main light period before dark. With lights off, HMG1 is functioning in dark-activated transcription, while CO is activating some genes that will stimulate FT. During the dark, other genes are activated, such as FT, which may represent the primary activated factor itself, or its target. This model accounts for both an hourglass timing mechanism (decay of certain Pfr pools) as well as an oscillating endogenous rhythm mechanism through control of gene expression. Finally in Pharbitis, phyA appears to have been eliminated as the major phytochrome pool candidate, whereas phyB, phyD, and phyE have not been eliminated as candidates for those phytochrome(s) possibly important in photoperiodic floral induction.
Conclusion
After nearly 100 years of research on the photoperiodic induction of flowering, with a large portion of it using Pharbitis nil as a model SDP, we are fast gaining an understanding of the key components of the underlying molecular mechanism in nonmodel systems. Some of the key molecular players have been identified, and those discussed specifically here include PHY, CO, and FT, as well as others not discussed here. Clearly, a great deal more molecular and biochemical research is needed to fully elucidate the mechanism of photoperiodic induction. For example, a detailed examination of the phytochrome proteins themselves and a detailed study of the effect of NB on the PHY genes and proteins would provide important and comprehensive information about the role of the phytochromes in photoperiodic flowering. Overall, considerable progress has been made, and we are finally beginning to understand the complex molecular events that require darkness in SDPs and light in LDPs and that depend on the PHY genes for photoperception at the beginning of the process and on FT for flower signaling at the end.
FOOTNOTES
1 The authors are thankful for the Professor Grady Webster Memorial Fund and its generous support of this research. The authors thank Professor Abraham H. Halevy (Hebrew University of Jerusalem, Israel) and Dr. A. Kadman-Zahavi (ARO, Volcani Center, Israel) for stimulating discussions about Pharbitis nil and photoperiodic floral induction. They also thank the two anonymous reviewers for helpful comments that improved this article. This research was supported by National Science Foundation Grant No. IOB 97-17249 (Developmental Systems) to S.O’N. 
4 Present address: College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018 PR China
5 Author for correspondence (e-mail: sdoneill{at}ucdavis.edu)
LITERATURE CITED
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, AND D. J. Lipman. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403–410.[CrossRef][Web of Science][Medline]
An, H., C. Roussot, P. Súarez-López, L. Corbesier, C. Vincent, M. Piñeiro, S. Hepsworth et al.. 2004. CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 131: 3615–3626.
Ausín, I., C. Alonso-Blanco, AND J.-M. Martínez-Zapeter. 2005. Environmental control of flowering. International Journal of Developmental Biology 49: 689–705.[CrossRef][Web of Science][Medline]
Bagnall, D. J., R. W. King, G. C. Whitelam, M. T. Boylan, D. Wagner, AND P. H. Quail. 1995. Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh. Plant Physiology 108: 1495–1503.[Abstract]
Balasubramanian, S., S. Sureshkumar, T. P. Agrawai, T. P. Michael, C. Wessinger, J. N. Maloof, R. Clarck et al.. 2006. The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana. Nature Genetics 38: 711–715.[CrossRef][Web of Science][Medline]
Bernier, G. 1988. The control of floral evocation and morphogenesis. Annual Review of Plant Physiology and Plant Molecular Biology 39: 175–219.[CrossRef][Web of Science]
Bernier, G., J.-M. Kinet, AND R. M. Sachs. 1981. The physiology of flowering, vol. I, The initiation of flowers; vol. II, Transition to reproductive growth. CRC Press, Boca Raton, Florida, USA.
Borthwick, H. A., S. B. Hendricks, AND M. W. Parker. 1952. The reaction controlling floral initiation. Proceedings of the National Academy of Sciences, USA 38: 929–933.
Bünning, E. 1936. Die endogene tagesrhythmik als grundlage der photoperiodischen reaction. Berichte der Deutschen Botanischen Gesellschaft 54: 590–607.
Cantón, F. R., AND P. H. Quail. 1999. Both phyA and phyB mediate light imposed repression of PHYA gene expression in Arabidopsis. Plant Physiology 121: 1207–1215.
Carter, C. E., A. Szmidt-Jaworska, M. Hughes, B. Thomas, AND S. Jackson. 2000. Phytochrome regulation of phytochrome A mRNA levels in the model short-day plant Pharbitis nil. Journal of Experimental Botany 51: 703–711.
Chevaillier, P. 1993. PEST sequences in nuclear proteins. International Journal of Biochemistry 25: 479–482.[CrossRef][Web of Science][Medline]
Christensen, A. H., AND P. H. Quail. 1989. Structure and expression of a maize phytochrome-encoding gene. Gene 85: 381–390.[CrossRef][Web of Science][Medline]
Childs, K., F. R. Miller, M.-M. Cordonnier-Pratt, L. H. Pratt, P. W. Morgan, AND J. E. Mullet. 1997. The sorghum photoperiod sensitivity gene, Ma3, encodes a phytochrome B. Plant Physiology 113: 611–619.[Abstract]
Clack, T., S. Matthews, AND R. A. Sharrock. 1994. The phytochrome apoprotein family in Arabidopsis is encoded by five genes: The sequences and expression of PHYD and PHYE. Plant Molecular Biology 25: 413–427.[CrossRef][Web of Science][Medline]
Corbesier, L., AND G. Coupland. 2006. The quest for florigen: A recent review of recent progress. Journal of Experimental Botany 57: 3395–3403.
Corbesier, L., C. Vincent, S. Jang, F. Fornara, Q. Fan, I. Searle, A. Giakountis et al.. 2007. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316: 1030–1033.
Dehesh, K., J. Tepperman, A. H. Christensen, AND P. H. Quail. 1993. PhyB is evolutionarily conserved and constitutively expressed in rice seedling shoots. Plant Cell 5: 1081–1088.
Devlin, P. F., P. R. H. Robson, S. R. Patel, L. Goosey, R. A. Sharrock, AND G. C. Whitelam. 1999. Phytochrome D acts in the shade-avoidance syndrome in Arabidopsis by controlling elongation growth and flowering time. Plant Physiology 119: 909–915.
Devlin, P. S., S. R. Patel, AND G. C. Whitelam. 1998. Phytochrome E influences internode elongation and flowering time in Arabidopsis. Plant Cell 10: 1479–1487.
Devlin, P. S., S. B. Rood, D. E. Somers, P. H. Quail, AND G. C. Whitelam. 1992. Photophysiology of the elongated internode (ein) mutant of Brassica rapa: ein mutant lacks a detectable phytochrome B-like polypepetide. Plant Physiology 100: 1442–1447.
Donoghue, M. J., AND S. Mathews. 1998. Duplicate genes and the root of angiosperms, with an example using phytochrome sequences. Molecular Phylogenetics and Evolution 9: 489–500.[CrossRef][Web of Science][Medline]
Eckardt, N. A. 2007. Measuring daylength: Pharbitis takes a different approach. Plant Cell 19: 2968–2969.
Evans, L. T. 1969. The nature of flower induction. In L. Evans [ed.], The induction of flowering: Some case histories, 457–480. Cornell University Press, Ithaca, New York, USA.
Evans, L. T. 1993. The physiology of flowering—Paradigms lost and paradigms regained. Australian Journal of Plant Physiology 20: 655–660.[Web of Science]
Fowler, S., K. Lee, H. Onouchi, A. Samach, K. Richardson, K. Morris, G. Coupland, AND J. Putterill. 1999. GIGANTEA: A circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains. EMBO Journal 18: 4679–4688.[CrossRef][Web of Science][Medline]
Franklin, K. A., T. Allen, AND G. C. Whitelam. 2007. Phytochrome A is an irradiance-dependent red light sensor. Plant Journal 50: 108–117.[CrossRef][Web of Science][Medline]
Franklin, K. A., S. J. Davis, W. M. Stoddart, R. M. Vierstra, AND G. C. Whitelam. 2003. Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis. Plant Cell 15: 1981–1989.
Franklin, K. A., V. S. Larner, AND G. C. Whitelam. 2005. The signal transducing photoreceptors of plants. International Journal of Developmental Biology 49: 653–664.[CrossRef][Web of Science][Medline]
Fredericq, H. 1964. Conditions determining effects of far-red and red irradiations on flowering response of Pharbitis nil. Plant Physiology 39: 812–816.
Furuya, M. 1993. Phytochromes: Their molecular species, gene family and functions. Annual Review of Plant Physiology and Plant Molecular Biology 44: 617–645.[CrossRef][Web of Science]
Garner, W. W., AND H. A. Allard. 1920. Effect of the relative length of day and night and other factors of the environment on growth and reproduction in plants. Journal of Agricultural Research 18: 553–606.
Goto, N., T. Kumagai, AND M. Koornneef. 1991. Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant. Plant Physiology 83: 209–215.[CrossRef]
Halliday, K. J., M. Koorneef, AND G. C. Whitelam. 1994. Phytochrome B and at least one other phytochrome mediate the accelerated flowering response of Arabidopsis thaliana L. to low red/far-red ratio. Plant Physiology 104: 1311–1315.[Abstract]
Halliday, K. J., AND G. C. Whitelam. 2003. Changes in photoperiod or temperature alter the functional relationships between phytochromes and reveal roles for phyD and phyE. Plant Physiology 131: 1913–1920.
Hamner, K. C. 1958. The mechanism of photoperiodism in plants. In Photobiology: Proceedings of 19th Annual Biology Colloquium, 7–16. Oregon State Chapter of Phi Kappa Phi, Oregon State College, Corvallis, Oregon, USA.
Hamner, K. C., AND J. Bonner. 1938. Photoperiodism in relation to hormones as factors in floral initiation and development. Botanical Gazette 100: 388–431.
Hamner, K. C., AND A. Takimoto. 1964. Circadian rhythms and plant photoperiodism. American Naturalist 98: 295–322.[CrossRef][Web of Science]
Hauser, B. A., M. M. Cordonnier-Pratt, F. Daniel-Vedele, AND L. H. Pratt. 1995. The phytochrome gene family in tomato includes a novel subfamily. Plant Molecular Biology 29: 1143–1155.[CrossRef][Web of Science][Medline]
Hayama, R., B. Agashe, E. Luley, R. King, AND G. Coupland. 2007. A circadian rhythm set by dusk determines the expression of FT homologs and the short-day photoperiodic flowering response in Pharbitis. Plant Cell 19: 2988–3000.
Hayama, R., AND G. Coupland. 2004. The molecular basis of diversity in the photoperiodic flowering response of Arabidopsis and rice. Plant Physiology 135: 677–684.
Heide, O. M. 1977. Photoperiodism in higher plants: An interaction of phytochrome and circadian rhythms. Physiologia Plantarum 39: 25–32.
Hendricks, S. B. 1960. Rates of change of photochrome as an essential factor for determining photoperiodism in plants. Symposium Quantitative Biology 25: 245–248.
Hennig, L., W. M. Stoddart, M. Dieterle, G. C. Whitelam, AND E. Schäfer. 2002. Phytochrome E controls light-induced germination of Arabidopsis. Plant Physiology 128: 194–200.
Heyer, A., AND C. Gatz. 1992. Isolation and characterization of a cDNA-clone coding for potato type B phytochrome. Plant Molecular Biology 18: 535–544.[CrossRef][Web of Science][Medline]
Imaizumi, T., AND S. A. Kay. 2006. Photoperiodic control of flowering: Not only by coincidence. Trends in Plant Science 11: 550–558.[CrossRef][Web of Science][Medline]
Imamura, S. 1967. Physiology of flowering in Pharbitis nil. Japanese Society of Plant Physiologists, Tokyo, Japan.
Imamura, S., AND A. Takimoto. 1955. Photoperiodic responses in Japanese morning glory, Pharbitis nil Chois, a sensitive short day plant. Botanical Magazine of Tokyo 68: 235–241.
Ishikawa, R., S. Tamaki, N. Yokoi, N. Inagaki, T. Shinomura, M. Takano, AND K. Shimamoto. 2005. Suppression of the floral activator Hd3a is the principal cause of the night break effect in rice. Plant Cell 17: 3326–3336.
Izawa, T., T. Oikawa, N. Sugiyama, T. Tanisaki, M. Yano, AND K. Shimamoto. 2002. Phytochrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice. Genes & Development 16: 2006–2020.
Izawa, T., Y. Takahashi, AND M. Yano. 2003. Comparative biology comes into bloom: Genomic and genetic comparison of flowering pathways in rice and Arabidopsis. Current Opinion in Plant Biology 6: 113–120.[CrossRef][Web of Science][Medline]
Izawa, T., T. Oikawa, S. Tokutomi, K. Okuno, AND K. Shimamoto. 2000. Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant). Plant Journal 22: 391–399.[CrossRef][Web of Science][Medline]
Jofuku, K. D., AND R. B. Goldberg. 1988. Analysis of plant gene structure. In C. H. Shaw [ed.], Plant Molecular biology: A practical approach, 37–66. IRL Press, Oxford, UK.
Johnson, E., M. Bradley, N. P. Harberd, AND G. C. Whitelam. 1994. Photoresponses of light-grown phyA mutants of Arabidopsis: Phytochrome A is required for the perception of daylength extensions. Plant Physiology 105: 141–149.[Abstract]
Kardailsky, I., V. K. Shukla, J. H. Ahn, N. Dagenais, S. K. Christensen, J. T. Nguyen, J. Chory et al.. 1999. Activation tagging of the floral inducer, FT. Science 286: 1962–1965.
Kay, S. A., B. Keith, K. Shinozaki, M.-L. Chyle, AND N.-H. Chua. 1989. The rice phytochrome gene: structure, autoregulated expression and binding of GT-1 to a conserved site in the 5' upstream region. Plant Cell 1: 351–360.
Kim, S.-J., J. Moon, I. Lee, J. Maeng, AND S.-R. Kim. 2003. Molecular cloning and expression analysis of a CONSTANS homologue, PnCOL1, from Pharbitis nil. Journal of Experimental Botany 54: 1879–1887.
King, R. W., D. Vince-Prue, AND P. H. Quail. 1978. Light requirements, phytochrome and photoperiodic induction of flowering of Pharbitis nil Chois. III. A comparison of spectrophotometric and physiological assay of phytochrome phototransformation during induction. Planta 141: 15–22.[CrossRef][Web of Science]
Kobayashi, Y., H. Kaya, K. Goto, M. Iwabuchi, AND T. Araki. 1999. A pair of related genes with antagonistic roles in mediating flowering signals. Science 286: 1960–1962.
Kobayashi, Y., AND D. Weigel. 2008. Move on up, it’s time for change—Mobile signals controlling photoperiod-dependent flowering. Genes & Development 21: 2371–2384.[Web of Science]
Kojima, S., Y. Takahashi, Y. Kobayashi, L. Monna, T. Sasaki, T. Araki, AND M. Yano. 2002. Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions. Plant & Cell Physiology 43: 1096–1105.
Lang, A. 1965. Physiology of flower initiation. In W. Ruhland [ed.], Encyclopedia of plant physiology, vol. XV, part I, 1380–1536. Springer-Verlag, New York., New York, USA.
Leopold, A. C. 1949. Flower initiation in total darkness. Plant Physiology 24: 530–533.
Li, L., AND J. C. Lagarias. 1994. Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, USA 91: 12535–12539.
Liu, J., J. Yu, L. McIntosh, H. Kende, AND J. A. D. Zeevaart. 2001. Isolation of a CONSTANS ortholog from Pharbitis nil and its role in flowering. Plant Physiology 125: 1821–1830.
Lumsden, P. J. 1991. Circadian rhythms and phytochrome. Annual Review of Plant Physiology and Plant Molecular Biology 42: 351–371.[CrossRef][Web of Science]
Lumsden, P. J., AND M. Furuya. 1986. Evidence for two actions of light in the photoperiodic induction of flowering in Pharbitis nil. Plant & Cell Physiology 17: 1541–1551.
Lumsden, P. J., B. Thomas, AND D. Vince-Prue. 1982. Photoperiodic control of flowering in dark-grown seedlings of Pharbitis nil Choisy. The effect of skeleton and continuous light photoperiods. Plant Physiology 70: 277–282.
Lumsden, P. J., AND D. Vince-Prue. 1984. The perception of dusk signals in photoperiod time-measurement. Plant Physiology 60: 427–432.[CrossRef]
Lumsden, P. J., D. Vince-Prue, AND M. Furuya. 1986. Phase shifting of the photoperiodic flowering response rhythm in Pharbitis nil by red light pulse. Physiologia Plantarum 67: 604–607.[CrossRef]
Lutcke, H. A., K. C. Chow, F. S. Mickel, K. A. Moss, H. F. Kern, AND G. A. Scheele. 1987. Selection of AUG initiation codons differs in plants and animals. EMBO Journal 6: 43–48.[Web of Science][Medline]
Mathews, S., AND R. A. Sharrock. 1997. Phytochrome gene diversity. Plant, Cell & Environment 20: 666–671.[CrossRef]
McClung, C. R. 2006. Plant circadian rhythms. Plant Cell 18: 792–803.
McCormac, A. C., G. C. Whitelam, M. T. Boylan, P. H. Quail, AND H. Smith. 1992. Contrasting responses of etiolated and light-adapted seedlings to red:far-red ratio: A comparison of wild type, mutant and transgenic plants has revealed differential functions of members of the phytochrome family. Journal of Plant Physiology 140: 707–714.[Web of Science]
Monte, E., J. M. Alonso, J. R. Ecker, Y. Zhang, J. Li, J. Young, S. Austin-Philips, AND P. H. Quail. 2003. Isolation and characterization of phyC mutants in Arabidopsi sreveals complex crosstalk between phytochrome signaling pathways. Plant Cell 15: 1962–1980.
Mouradov, A., F. Cremer, AND G. Coupland. 2002. Control of flowering time: Interacting pathways as a basis for diversity. Plant Cell 14: S111–S130.
Nagatani, A., J. Chory, AND M. Furuya. 1991. Phytochrome B is not detectable in the hy3 mutant of Arabidopsis, which is deficient in responding to end-of-day far-red treatments. Plant & Cell Physiology 32: 1119–1122.
Nagatani, A., J. W. Reed, AND J. Chory. 1993. Isolation and initial characterization of Arabidopsis mutants that are deficient in phytochrome A. Plant Physiology 102: 269–277.[Abstract]
O’Neill, S. D. 1992. The photoperiodic control of flowering: Progress towards understanding the mechanism of induction. In W. J. VanDerWoude, and S. J. Britz [eds.], Photomorphogenesis in plants. Emerging strategies for crop improvement. Photochemistry and Photobiology 56: 789–801.[CrossRef][Web of Science]
O’Neill, S. D. 1993. Changes in gene expression associated with floral induction and evocation. In B. R. Jordan [ed.], The molecular biology of flowering, 69–92. CAB International, Wallingford, UK.
O’Neill, S. D., AND C. C. Zheng. 1998. Abundance of mRNAs encoding HMG1/HMGs class high-mobility-group DNA-binding proteins is differentially regulated in cotyledons of Pharbitis nil. Plant Molecular Biology 37: 235–241.[CrossRef][Web of Science][Medline]
O’Neill, S. D., X. S. Zhang, AND C. C. Zheng. 1994. Dark and circadian regulation of mRNA accumulation in the short-day plant Pharbitis nil. Plant Physiology 104: 569–580.[Abstract]
Paraska, J. R., AND C. Spector. 1974. The initiation of an endogenous rhythm affecting flower bud formation in Pharbitis nil. Plant Physiology 32: 62–65.[CrossRef]
Park, D. H., D. E. Somers, Y. S. Kim, Y. H. Choy, H. K. Lim, M. S. Soh, H. J. Kim et al.. 1999. Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285: 1579–1582.
Parks, B. M., AND P. H. Quail. 1993. hy8, a new class of Arabidopsis long hypocotyl mutants deficient in functional phytochrome A. Plant Cell 5: 39–48.
Perilleux, C., AND G. Bernier. 2002. The control of flowering: Do genetic and physiological approaches converge? In S. D. O’Neill, and J. A. Roberts [eds.], Plant reproduction. Annual plant reviews, vol. 6, 1–32. Sheffield Academic Press, Sheffield, UK.
Putterill, J., F. Robson, K. Lee, R. Simon, AND G. Coupland. 1995. The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80: 847–857.[CrossRef][Web of Science][Medline]
Quail, P. H. 1991. Phytochrome A light-activated molecular switch that regulates plant gene expression. Annual Review of Genetics 25: 389–409.[CrossRef][Web of Science][Medline]
Quail, P. H. 2002. Phytochrome photosensory signaling networks. Nature Reviews Molecular Cell Biology 3: 85–93.[CrossRef][Web of Science][Medline]
Reed, J. W., P. Nagpal, D. S. Poole, M. Furaya, AND J. Chory. 1993. Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5: 147–157.[Abstract]
Rogers, S., R. Wells, AND M. Reichsteiner. 1986. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 234: 364–368.
Saji, H., D. Vince-Prue, AND M. Furuya. 1983. Studies on the photoreceptors for the promotion and inhibition of flowering of dark-grown seedlings of Pharbitis nil Choisy. Plant & Cell Physiology 25: 1183–1189.
Salisbury, F. B. 1982. Photoperiodism. Horticultural Reviews 4: 66–105.
Samach, A., H. Onouchi, S. E. Gold, G. S. Ditta, Z. Schwarz-Sommer, M. F. Yanofsky, AND G. Coupland. 2000. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288: 1613–1616.
Sambrook, J., E. F. Fritsch, AND T. Maniatis. 1989. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
Sanger, F., S. Nicklen, AND A. R. Coulson. 1977. DNA sequencing with chain-termination inhibitors. Proceedings of the National Academy of Sciences, USA 74: 5463–5467.
Sharrock, R. A., AND P. H. Quail. 1989. Novel phytochrome sequences in Arabidopsis thaliana: Structure, evolution, and differential expression of a plant regulatory photoreceptor family. Genes & Development 3: 1745–1757.
Silverthorne, J., AND E. M. Tobin. 1984. Demonstration of transcriptional regulation of specific genes by phytochrome action. Proceedings of the National Academy of Sciences, USA 81: 1112–1116.
Smith, H., AND G. C. Whitelam. 1990. Phytochrome, a family of photoreceptors with multiple physiological roles. Plant, Cell & Environment 13: 695–707.[CrossRef]
Spector, C., AND J. R. Paraska. 1973. Rhythmicity of flowering in Pharbitis nil. Physiologia Plantarum 29: 402–405.[CrossRef]
Súarez-López, P., K. Wheatley, F. Robson, H. Onouchi, F. Valverde, AND G. Coupland. 2001. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature 410: 1116–1120.[CrossRef][Medline]
Sugiyama, N., T. Izawa, T. Oikawa, AND K. Shimamoto. 2001. Light regulation of circadian clock-controlled gene expression in rice. Plant Journal 26: 607–615.[CrossRef][Web of Science][Medline]
Sullivan, J. A., AND X. W. Deng. 2003. From seed to seed: The role of photoreceptors in Arabidopsis development. Developmental Biology 260: 289–297.[CrossRef][Web of Science][Medline]
Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sinauer, Sunderland, Massachusetts, USA.
Szmidt-Jaworska, A., K. Jaworski, AND J. Kopcewicz. 2000. The level of phyA in Pharbitis nil Choisy during the photoperiodic flower induction. Acta Societatis Botanicorum Poloniae 69: 269–272.[Web of Science]
Takano, M., N. Inagaki, X. Xie, N. Yuzurihara, F. Hhihara, T. Ishizhka, M. Yano, M. Nishimura, A. Miyao, H. Hirochika, AND T. Shinomura. 2005. Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice. Plant Cell 7: 3311–3325.
Takimoto, A. 1967. Pharbitis nil Choisy. In L. T. Evans [ed.], The induction of flowering: Some case histories, 90–115. Cornell University Press, Ithaca, New York, USA.
Takimoto, A., AND K. C. Hamner. 1965. Studies on red light interruption in relation to timing mechanisms in the photoperiodic response of Pharbitis nil. Plant Physiology 40: 852–854.
Takimoto, A., AND K. Ikeda. 1960. Studies on the light controlling flower initiation of Pharbitis nil. VII. Light-break. Botanical Magazine of Tokyo 37: 341–347.
Takimoto, A., AND H. Saji. 1984. A role of phytochrome in photoperiodic induction: Two-phytochrome pool theory. Physiologia Plantarum 61: 675–682.[CrossRef]
Thomas, B. 1991. Phytochrome and photoperiodic induction. Physiologia Plantarum 81: 571–577.[CrossRef]
Thomas, B. 2006. Light signals and flowering. Journal of Experimental Botany 57: 3387–3393.
Thompson, J., T. J. Gibson, F. Plewniak, F. Jeanmougin, AND D. G. Higgins. 1997. The Clustal_X window interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.
Thummler, F., M. Dufner, P. Kreisl, AND P. Dittrich. 1992. Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated kinase. Plant Molecular Biology 20: 1003–1017.[CrossRef][Web of Science][Medline]
Tokuhisa, J. G., S. M. Daniels, AND P. H. Quail. 1985. Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L. Planta 164: 321–332.[CrossRef][Web of Science]
Valverde, F., A. Mouradov, W. Soppe, D. Ravenscroft, A. Samach, AND G. Coupland. 2004. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303: 1003–1006.
Vince-Prue, D. 1975. Photoperiodism in plants. McGraw Hill, London, UK.
Vince-Prue, D. 1986. The duration of light and photoperiodic responses. In R. E. Kendrick, and G. H. M. Kroneberg [eds.], Photomorphogenesis in plants, 269–305. Martinus Nijhoff, Dordrecht, Netherlands.
Vince-Prue, D. 1989. The role of phytochrome in the control of flowering. Flowering Newsletter 8: 54–57.
Vince-Prue, D., AND J. Gressel. 1985. Pharbitis nil. En. Japanese morning glory. In A. H. Halevy [ed.], Handbook of flowering, vol. IV, 47–81. CRC Press, Boca Raton, Florida, USA.
Vince-Prue, D., AND P. J. Lumsden. 1987. Inductive events in leaves: Time measurement and photoperception in the short-day plant, Pharbitis nil. In J. G. Atherton [ed.], Manipulation of flowering, 255–268. Butterworth Press, London,UK.
Whitelam, G. C., E. Johnson, J. Peng, P. Carol, M. L. Anderson, J. S. Cowl, AND N. P. Harberd. 1993. Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light. Plant Cell 5: 757–768.
Yano, M., Y. Katayose, M. Ashikara, U. Yamanouchi, L. Monna, T. Fuse, T. Baba, K. Yamamoto, Y. Umehara, Y. Nagamura, AND T. Sasaki. 2000. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 12: 2473–2484.
Yano, M., S. Kojima, Y. Takahashi, H. Lin, AND T. Sasaki. 2001. Genetic control of flowering time in rice, a short-day plant. Plant Physiology 127: 1425–1429.
Yanovsky, M. J., AND S. A. Kay. 2002. Molecular basis of seasonal time measurement in Arabidopsis. Nature 419: 308–312.[CrossRef][Medline]
Zeevaart, J. A. D. 1976. Physiology of flower formation. Annual Review of Plant Physiology 27: 321–384.[Web of Science]
Zeevaart, J. A. D. 2006. Florigen coming of age after 70 years. Plant Cell 18: 1783–1789.
Zheng, C. C., A. Q. Bui, AND S. D. O’Neill. 1993. Abundance of an mRNA encoding a high mobility group DNA-binding protein is regulated by light and an endogenous rhythm. Plant Molecular Biology 23: 813–823.[CrossRef][Web of Science][Medline]
Zheng, C. C., R. D. Porat, P. Lu, AND S. D. O’Neill. 1998. PNZIP is a novel mesophyll-specific cDNA that is regulated by phytochrome and a circadian rhythm and encodes a protein with a leucine zipper motif. Plant Physiology 116: 27–35.
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