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Genetic Linkages Between Circadian Clock-Associated Components and Phytochrome-Dependent Red Light Signal Transduction in Arabidopsis thaliana

Genetic Linkages Between Circadian Clock-Associated Components and Phytochrome-Dependent Red... Abstract The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB. Introduction For higher plants, light is one of the most prominent environmental signals and it regulates almost all aspects of developmental stages, as well as physiological states (for a review, see Chen et al. 2004). The thorough understanding of light-mediated signal transduction pathways is one of the major current paradigms of plant biology. A number of studies have been carrried out with reference to the mechanisms underlying such light signal transduction pathways in many plant species. By employing the model higher plant Arabidopsis thaliana, here we focus on an instance of the red light signaling pathway that especially regulates the elongation of hypocotyls during early photomorphogenesis, with special reference to its close linkage with the circadian clock. In A. thaliana, circadian rhythms are extremely relevant to a wide range of biological processes, including diurnal changes in photosynthetic activity and photoperiodic control of flowering time (McClung 2000, Mouradov et al. 2002, Eriksson and Millar 2003, Yanovsky and Kay 2003, Salome and McClung 2004, Nozue and Maloof 2006). The results of recent intensive studies have begun to shed light on the molecular mechanisms underlying the central clock (for recent reviews, see Gardner et al. 2006, Mizuno and Nakamichi 2005, McClung 2006). The current best candidates for such clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its partially redundant homolog LHY (LATE ELONGATED HYPOCOTYL), both of which are single MYB-containing transcription factors (Schaffer et al. 1998, Wang and Tobin 1998, Alabadi et al. 2002, Mizoguchi et al. 2002). In addition, five members of the small PRR (PSEUDO-RESPONSE REGULATOR) family, i.e. PRR1, PRR3, PRR5, PRR7 and PRR9, are believed to be another type of clock-associated component (for recent papers, see Farre et al. 2005, Nakamichi et al. 2005a, Nakamichi et al, 2005b, Salome and McClung 2005). The first described representative of PRRs is TOC1 (TIMING OF CAB EXPRESSION) 1, which is identical to PRR1 (Somers et al. 1998, Strayer et al. 2000) [thus referred to as TOC1 (PRR1) in this text, when necessary for emphasis]. It was proposed that these two types of clock-associated genes [CCA1/LHY and TOC1 (PRR1)] form a negative–positive transcriptional feedback loop that generates fundamental circadian rhythms (Alabadi et al. 2001, see also Fig. 8 later). Subsequently, many other clock-associated genes have been identified and incorporated into the single-loop model, and currently somewhat complicated interlocking multiloop models are favorably envisaged (for reviews, see Mizuno and Nakamichi 2005, Gardner et al. 2006, McClung 2006). Furthermore, a consistent multiloop clock model has recently been built through mathematical simulation (Locke et al. 2006, Zeilinger et al. 2006). During the course of these intensive studies, it was recognized that plants carrying a lesion (i.e. loss of function or misexpression) in a given clock-associated gene commonly display a light response-associated phenotype (Dowson-Day and Millar 1999). Under standard laboratory conditions, the length of hypocotyls of seedlings is finely regulated by a given fluence rate of red light during early photomorphogenesis. This event appears to be closely relevant to an aspect of natural shade avoidance (Nozue and Maloof 2006). In this respect, many circadian-associated mutants and transgenic lines, if not all, commonly show characteristic responses (i.e. hyper- or hyposensitivity) to a wide range of red light fluence rates (Sato et al. 2002, Eriksson et al. 2003, Kaczorowski and Quail 2003, Khanna et al. 2003, Mas et al. 2003, Yamamoto et al. 2003, Fujimori et al. 2004, Fujimori et al. 2005, Mizoguchi et al. 2005). For instance, a cca1 lhy double loss-of-function mutant is hypersensitive to red light, resulting in a phenotype of short hypocotyls, whereas a toc1 hyopomorphic mutant is hyposensitive, resulting in a phenotype of long hypocotyls. In other words, there is a close linkage between the regulation of hypocotyl elongation during early photomorphogenesis and circadian clock-associated components. Nevertheless, little is known about the molecular bases underlying such an intriguing linkage. Here we address this particular issue by employing a large number of Arabidopsis mutants and transgenic lines of a set of clock-associated genes, including CCA1/LHY and PRRs (see Table 1). Furthermore, we also employed certain mutant alleles (including phyA, phyB, pif3 and pil6), which appear to be directly relevant to red light signal transduction per se (see Table 1). Arabidopsis thaliana has five homologous red light photoreceptors, or phytochromes (designated as phyA–phyE) (for reviews, see Casal and Yanovsky 2005, Franklin et al. 2005). Among these red light photoreceptors, phyA and phyB appear to play major roles. Such phyA- and phyB-dependent signal transduction pathways have been characterized intensively at the molecular level (for a review, see Quail 2002). To clarify downstream signaling pathways, many phytochrome-interacting proteins have also been identified. Among such transcription factors, a set of basic helix–loop–helix (bHLH) transcription factors is best characterized, and they have been collectively designated as PIF/PIL (PHYTOCHROME-INTERACTING FACTOR/LIKE) proteins (for a review, see Duek and Fankhauser 2005). Representative is PIF3; this transcription factor was extensively characterized in terms of phytochrome-mediated immediate-early signal transduction (Bauer et al. 2004, Monte et al. 2004). The homologous PIL6 is especially interesting in that its expression is under the control of circadian rhythm (Yamashino et al. 2003). Importantly, both PIF3 and PIL6 are implicated in the early photomorphogenesis of young seedlings under red light (Fujimori et al. 2004). Table 1 List of Arabidopsis mutants and/or transgenic lines characterized in this study Mutants and/or transgenic linesa  Background  Red light sensitivity (degree of IEH)b  References  PRR1-ox  Col  Hyper (+++++)  Makino et al. (2002); Sato et al. (2002)  PRR1-ox prr5-11  Col  Hyper (+++++)  This study  PRR5-ox  Col  Hyper (+++++)  Sato et al. (2002)  PRR5-ox toc1-2  C24  Hyper (++++)  This study  PRR7-ox  Col  Hyper (++++)  Matsushika et al. (2007b)  PRR9-ox  Col  Hyper (++++)  Sato et al. (2002)  cca1-1 lhy11  Col  Hyper (+++)  Mizoguchi et al. (2002)  cca1-1 lhy11 prr7-11  Col  Hyper (++)  Nakamichi et al. (2007)  cca1-1 lhy11 prr5-11  Col  Hyper (++)  Nakamichi et al. (2007)  pif3-1  Col  Hyper (++)  Kim et al. (2003)  pil6-1  Col  Hyper (++)  Fujimori et al. (2004)  lhy11  Col  Hyper (++)  Mizoguchi et al. (2002)  cca1-1  Col  Hyper (++)  Green and Tobin (1999)  lhy11 prr5-1  Col  Hyper (+)  Nakamichi et al. (2007)  cca1-1 lhy11 prr7-11 prr5-11  Col  ±  Nakamichi et al. (2007)  Wild type  Col  ±  Nakamichi et al. (2007)  PRR1-ox phyA-201 phyB-5  Ler  ±  This study  prr5-11 pil3-1  Col  ±  This study  prr5-11 pil6-1  Col  ±  This study  prr9-10  Col  Hypo (−)  Ito et al. (2003)c  cca1-1 prr5-11  Col  Hypo (−)  Nakamichi et al. (2007)  cca1-1 prr7-11  Col  Hypo (−)  Nakamichi et al. (2007)  PRR3-ox  Col  Hypo (− −)  Murakami et al. (2004)  prr5-11  Col  Hypo (− − −)  Yamamoto et al. (2003)d  CCA1-ox PRR5-ox  Col  Hypo (− − −)  Fujimori et al. (2005)  prr9-10 prr5-11  Col  Hypo (− − −)  Nakamichi et al. (2005b)  lhy11 prr7-11  Col  Hypo (− − −)  Nakamichi et al. (2007)  prr7-11  Col  Hypo (− − −)  Yamamoto et al. (2003)e  prr7-11 pil6-1  Col  Hypo (− − −)  This study  prr7-11 pif3-1  Col  Hypo (− − −)  This study  CCA1-ox  Col  Hypo (− − − −)  Wang and Tobin (1998); Matsushika et al. (2002b)  toc1-2  C24 Col  Hypo (− − − −) Hypo (− − − −)  Mas et al. (2003); This studyc  cca1-1 toc1-2  Col  Hypo (− − − −)  This study  lhy11 toc1-2  Col  Hypo (− − − −)  This study  cca1-1 lhy11 toc1-2  Col  Hypo (− − − −)  This study  prr9-10 prr7-11  Col  Hypo (− − − −)  Farre et al. (2005); Salome and McClung (2005); Nakamichi et al. (2005b)  cca1-1 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  lhy11 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2005a)  PIL6-ox  Col  Hypo (− − − − −)  Fujimori et al. (2004)  prr9-10 prr7-11 prr5-11  Col  Hypo (− − − − −)  Nakamichi et al. (2005b)  phyB-9 pil6-1  Col  Hypo (− − − − −)  This study  phyB-9 pif3-1  Col  Hypo (− − − − −)  This study  phyB-9  Col  Hypo (− − − − −)  Reed et al. (1993)  phyB-9 prr7-11  Col  Hypo (− − − − −)  This study  phyB-9 prr7-11 prr5-11  Col  Hypo (− − − − −)  This study  PRR5-ox phyA-201 phyB-5  Ler  Hypo (− − − − −)  This study  phyA-201 phyB-5  Ler  Hypo (− − − − −)  Reed et al. (1994)  Mutants and/or transgenic linesa  Background  Red light sensitivity (degree of IEH)b  References  PRR1-ox  Col  Hyper (+++++)  Makino et al. (2002); Sato et al. (2002)  PRR1-ox prr5-11  Col  Hyper (+++++)  This study  PRR5-ox  Col  Hyper (+++++)  Sato et al. (2002)  PRR5-ox toc1-2  C24  Hyper (++++)  This study  PRR7-ox  Col  Hyper (++++)  Matsushika et al. (2007b)  PRR9-ox  Col  Hyper (++++)  Sato et al. (2002)  cca1-1 lhy11  Col  Hyper (+++)  Mizoguchi et al. (2002)  cca1-1 lhy11 prr7-11  Col  Hyper (++)  Nakamichi et al. (2007)  cca1-1 lhy11 prr5-11  Col  Hyper (++)  Nakamichi et al. (2007)  pif3-1  Col  Hyper (++)  Kim et al. (2003)  pil6-1  Col  Hyper (++)  Fujimori et al. (2004)  lhy11  Col  Hyper (++)  Mizoguchi et al. (2002)  cca1-1  Col  Hyper (++)  Green and Tobin (1999)  lhy11 prr5-1  Col  Hyper (+)  Nakamichi et al. (2007)  cca1-1 lhy11 prr7-11 prr5-11  Col  ±  Nakamichi et al. (2007)  Wild type  Col  ±  Nakamichi et al. (2007)  PRR1-ox phyA-201 phyB-5  Ler  ±  This study  prr5-11 pil3-1  Col  ±  This study  prr5-11 pil6-1  Col  ±  This study  prr9-10  Col  Hypo (−)  Ito et al. (2003)c  cca1-1 prr5-11  Col  Hypo (−)  Nakamichi et al. (2007)  cca1-1 prr7-11  Col  Hypo (−)  Nakamichi et al. (2007)  PRR3-ox  Col  Hypo (− −)  Murakami et al. (2004)  prr5-11  Col  Hypo (− − −)  Yamamoto et al. (2003)d  CCA1-ox PRR5-ox  Col  Hypo (− − −)  Fujimori et al. (2005)  prr9-10 prr5-11  Col  Hypo (− − −)  Nakamichi et al. (2005b)  lhy11 prr7-11  Col  Hypo (− − −)  Nakamichi et al. (2007)  prr7-11  Col  Hypo (− − −)  Yamamoto et al. (2003)e  prr7-11 pil6-1  Col  Hypo (− − −)  This study  prr7-11 pif3-1  Col  Hypo (− − −)  This study  CCA1-ox  Col  Hypo (− − − −)  Wang and Tobin (1998); Matsushika et al. (2002b)  toc1-2  C24 Col  Hypo (− − − −) Hypo (− − − −)  Mas et al. (2003); This studyc  cca1-1 toc1-2  Col  Hypo (− − − −)  This study  lhy11 toc1-2  Col  Hypo (− − − −)  This study  cca1-1 lhy11 toc1-2  Col  Hypo (− − − −)  This study  prr9-10 prr7-11  Col  Hypo (− − − −)  Farre et al. (2005); Salome and McClung (2005); Nakamichi et al. (2005b)  cca1-1 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  lhy11 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2005a)  PIL6-ox  Col  Hypo (− − − − −)  Fujimori et al. (2004)  prr9-10 prr7-11 prr5-11  Col  Hypo (− − − − −)  Nakamichi et al. (2005b)  phyB-9 pil6-1  Col  Hypo (− − − − −)  This study  phyB-9 pif3-1  Col  Hypo (− − − − −)  This study  phyB-9  Col  Hypo (− − − − −)  Reed et al. (1993)  phyB-9 prr7-11  Col  Hypo (− − − − −)  This study  phyB-9 prr7-11 prr5-11  Col  Hypo (− − − − −)  This study  PRR5-ox phyA-201 phyB-5  Ler  Hypo (− − − − −)  This study  phyA-201 phyB-5  Ler  Hypo (− − − − −)  Reed et al. (1994)  aFor the original reports with regard to these mutant alleles and/or transgenic lines, see the references cited. In this text, however, note also that they were designated simply without the allele numbers, solely for clarity of the text. bThe sensitivity to red light in terms of IEH (inhibition of elongation of hypocotyls) was compared relative to each other. When seedlings with a certain genetic background showed very short hypocotyls at a given fluence rate of red light, this was scored as ‘hyper-sensitive (+++++)’, whereas when they showed very long hypocotyls, this was scored as ‘hypo-sensitive (− − − − −)’. In this sense, it should be noted that these relative values are semi-quantitative. For the corresponding quantitative data, however, see the results of Figs. 1–7, and the appropriate references. cOther groups have also characterized the same prr9 allele (designated as prr9-1, see Eriksson et al. 2003, Michael et al. 2003). dTwo other prr5 alleles (prr5-1 and prr5-3) were reported, which showed similar phenotypes to that shown here for prr5-11 (Eriksson et al. 2003, Salome and McClung 2005). eMichael et al. (2003) reported the same prr7 allele (as prr7-3). However, Kaczorowski and Quail (2003) reported another prr7 allele in a different background, consistently suggesting a link between PRR7 and the red light signal transduction. fIn this study, the original toc1-2 allele in C24 was transferred into Col by extensive introgression, in order to compare its phenotype consistently with others in the Col background (see Materials and Methods). It may be worth mentioning that the resulting toc1-2 mutant with the Col background showed the expected phenotypes: ‘short-period of free-running rhythms of certain clock-controlled genes’, and ‘markedly early flowering’, as well as ‘hypo-sensitiveness to red light’. It may be also noted that toc1-2 is a strong hypomorphic mutant in that the plants produce about 6% of wild-type transcripts (Strayer et al. 2000), but its short period phenotype is essentially the same as that of a toc1 loss-of function mutant, as far as the phenotype regarding circadian rhythm is concerned (Locke et al. 2006). View Large By employing a number of the clock- and phytochrome-associated mutants mentioned above, here we carried out an extensive series of combinatorial genetic and epistatic analyses, in order to clarify the linkage between the circadian clock and the red light signal transduction pathway. Based on these results, we propose a genetic model. Results Early photomorphogenesis of seedlings under red light in clock-associated mutants Seeds of A. thaliana were germinated and grown on MS gellan-gum plates at a given fluence rate of red light for 2 or 3 d. To examine the sensitivity to red light during early photomorphogenesis, the seedlings were scored with regard to their hypocotyl length in comparison with etiolated seedlings. The seedlings examined in this study were a set of clock-associated and red light signaling-associated mutants or transgenic lines, including a PRR1 (TOC1)-overexpressing (ox) transgenic line (designated PRR1-ox), a toc1 hypomorphic mutant, a PRR5-ox transgenic line, a prr7 prr5 double loss-of-function mutant, a CCA1-ox transgenic line, a cca1 lhy double loss-of-function mutant, a pif3 mutant, a pil6 mutant, a phyA phyB double mutant and others (see Table 1 for details, the allele number of each mutation was omitted for clarity in the text). To interpret the genetic data properly in the unified genetic Columbia (Col) background, the original toc1-2 allele from the C24 background (Strayer et al. 2000) and the cca1-1 lhy11 double mutant alleles from the Ler background (Mizoguchi et al. 2002) were introgressed into the Col background by back-crossing repeatedly (more than four times; see Materials and Methods). The results indicated that some of these mutant seedlings (pif3 mutant, cca1 lhy double mutant, PRR5-ox line and PRR1-ox line) are hypersensitive to red light, and others (CCA1-ox, prr7 prr5 double mutant and toc1 mutant) are hyposensitive compared with the wild-type Col seedlings (see Table 1). These observations are consistent with those reported previously by us and other researchers (Sato et al. 2002, Eriksson et al. 2003, Kaczorowski and Quail 2003, Kim et al. 2003, Mas et al. 2003, Yamamoto et al. 2003, Mizoguchi et al. 2005, Nakamichi et al. 2005a). The results consistently implicated that PIF3 and CCA1/LHY act in the phyA/phyB-mediated light-signal transduction pathway as negative regulators, and PRR7, PRR5 and TOC1 (PRR1) act as positive regulators. Based on these interpretations, the main aim of this study was the clarification of genetic linkage between the circadian clock-associated components and phytochrome-dependent red light signal transduction with special reference to early photomorphogenesis of seedlings. Photomorphogenesis of adult plants was not a concern of this study because such processes are very complicated, and are clearly distinct from the event considered in this study. Combinatorial genetics with regard to CCA1/LHY and TOC1 (PRR1) As mentioned above, toc1 seedlings display a phenotype of hyposensitivity to red light, whereas cca1 lhy double mutants show a phenotype of hypersensitivity (Fig. 1A). Thus, these are ideal mutant alleles for epistasis analysis. Hence, a homozygous cca1 lhy toc1 triple mutant line was established. The sensitivity to red light of seedlings was examined compared with those of appropriate references. Similarly to the toc1 single mutant, the cca1 lhy toc1 triple mutant seedlings showed a phenotype of long hypocotyls under the conditions tested (red light, 0.5 μmol m−2 s−1) (Fig. 1A). This result was confirmed under a wide range of fluence rates of red light (Fig. 1B). Although the results were less evident at a given low fluence rate (0.05 μmol m−2 s−1), it was clearly indicated that the cca1 lhy toc1 triple mutant (2-day-old seedlings) did indeed show a phenotype of long hypocotyls at both 0.5 and 5 μmol m−2 s−1. These observations were confirmed by conducting another independent experiment with 3-day-old seedlings (Fig. 1C). Hence, we concluded that the toc1 lesion is epistatic to the cca1 lhy double mutations. Fig. 1 View largeDownload slide Red light responses of the cca1 lhy toc1 triple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) The indicated set of clock-associated mutants was examined. They were grown under a given fluence rate (0.5 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) on MS plates for 2 d. Among many seedlings examined (n > 25), two representatives were photographed for each. (B, C) Fluence rate response curves regarding the length of hypocotyls. Two independent experiments (biological replicates, experiment 1 and experiment 2) were carried out with essentially the same results. These data are presented here. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of continuous red light for 2 d (Exp. 1) or for 3 d (Exp. 2). Error bars represent SD values. Fig. 1 View largeDownload slide Red light responses of the cca1 lhy toc1 triple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) The indicated set of clock-associated mutants was examined. They were grown under a given fluence rate (0.5 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) on MS plates for 2 d. Among many seedlings examined (n > 25), two representatives were photographed for each. (B, C) Fluence rate response curves regarding the length of hypocotyls. Two independent experiments (biological replicates, experiment 1 and experiment 2) were carried out with essentially the same results. These data are presented here. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of continuous red light for 2 d (Exp. 1) or for 3 d (Exp. 2). Error bars represent SD values. To extend these genetic analyses, we established a series of homozygous mutants with every possible combination with regard to the three mutations (cca1, lhy and toc1). These mutants (seven types) together with Col were examined in terms of red light sensitivity at a given fluence rate (Fig. 2A, B). The results were consistent with the view that the toc1 mutation is fully epistatic to both the cca1 and lhy mutations. The results also suggested that the functions of CCA1 and LHY are partially (but not completely) redundant in this respect. These results are consistent with the assumption that the CCA1/LHY homologous components negatively regulate a red light signal transduction pathway through the function of TOC1 (PRR1), which acts downstream as a positive regulator of the pathway. Fig. 2 View largeDownload slide Red light responses of a set of homozygous mutants with every possible combination of three mutant alleles (cca1 lhy toc1). (A) The seedlings (seven types of mutants together with Col) were grown under a given fluence rate of continuous red light (5 μmol m−2 s−1) and in the dark for 3 d. (B) The resulting lengths of their hypocotyls were quantitatively examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (0.5 and 5 μmol m−2 s−1), and similar results were obtained (data not shown). Fig. 2 View largeDownload slide Red light responses of a set of homozygous mutants with every possible combination of three mutant alleles (cca1 lhy toc1). (A) The seedlings (seven types of mutants together with Col) were grown under a given fluence rate of continuous red light (5 μmol m−2 s−1) and in the dark for 3 d. (B) The resulting lengths of their hypocotyls were quantitatively examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (0.5 and 5 μmol m−2 s−1), and similar results were obtained (data not shown). Combinatorial genetics with regard to CCA1/LHY and PRR7/PRR5 As mentioned above, cca1 lhy double mutant seedlings display a phenotype of short hypocotyls under a given fluence rate of red light, while the prr7 prr5 double mutant shows a phenotype of long hypocotyls under the same conditions (Table 1). Hence, these are also ideal mutant alleles for epistasis analyses. This was done by extensively crossing cca1 lhy and prr7 prr5 double mutants (for details, see Nakamichi et al. 2007). The phenotype of isolated homozygous ccal lhy prr7 prr5 quadruple mutant seedlings was first examined under a given fluence rate of red light (Fig. 3A, 10 μmol m−2 s−1). The quadruple mutant seedlings showed moderate sensitivity to red light, similar to wild-type Col seedlings. These results were confirmed by examining the phenotype under a wide range of fluence rates of red light (Fig. 3B). Within the range of red light fluence rates tested (1 and 10 μmol m−2 s−1), the quadruple mutant seedlings showed a phenotype of moderate hypocotyl length that was similar to that of Col, although this phenotype was less clear at a low fluence rate (0.1 μmol m−2 s−1). These results suggested that both pairs of clock components (CCA1/LHY and PRR7/PRR5) regulate the length of hypocotyls in response to red light independently of (or in parallel with) each other. Importantly, these effects of CCA1/LHY and PRR7/PRR5 are antagonistic. This linkage between CCA1/LHY and PRR7/PRR5 is in sharp contrast to that observed between CCA1/LHY and TOC1 (PRR1) (compare the results of Figs. 1, 3). Fig. 3 View largeDownload slide Red light responses of the cca1 lhy prr7 prr5 quadruple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) For the first experiment (Exp. 1), the indicated set of mutant seedlings, together with the wild-type Col seedlings, was grown under a given fluence rate of continuous red light (10 μmol m−2 s−1) and in the dark for 2 d. The resulting lengths of their hypocotyls were quantitatively examined (n > 25). (B) To ensure reproducibility, the independent experiments were carried out (Exp. 2). In B, fluence rate response curves regarding the length of hypocotyls were examined, with results consistent with those in A. The seedlings were grown under a wide range of fluence rates of red light for 3 d. The resulting lengths of their hypocotyls were examined (n > 25). Error bars represent SD values. Fig. 3 View largeDownload slide Red light responses of the cca1 lhy prr7 prr5 quadruple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) For the first experiment (Exp. 1), the indicated set of mutant seedlings, together with the wild-type Col seedlings, was grown under a given fluence rate of continuous red light (10 μmol m−2 s−1) and in the dark for 2 d. The resulting lengths of their hypocotyls were quantitatively examined (n > 25). (B) To ensure reproducibility, the independent experiments were carried out (Exp. 2). In B, fluence rate response curves regarding the length of hypocotyls were examined, with results consistent with those in A. The seedlings were grown under a wide range of fluence rates of red light for 3 d. The resulting lengths of their hypocotyls were examined (n > 25). Error bars represent SD values. To extend these genetic analyses, we established a series of homozygous mutants with every possible combination with regard to the four mutations (cca1, lhy, prr7 and prr5) (for details, see Nakamichi et al. 2007). These mutants (15 types) together with Col were examined in terms of red light sensitivity at a given fluence rate (Fig. 4). The observed hypocotyl length of these mutants varied gradually from the shortest (the cca1 lhy double mutant) to the longest (the prr7 prr5 double mutant). In these experiments, it was rather difficult to determine statistically the exact degrees of phenotypes amongst these mutants relative to each other. However, it was clear that the cca1 lhy prr7 prr5 quadruple mutant showed a hypocotyl length similar to that of the wild type (Col). The results of these combinatorial epistasis analyses were fully consistent with the results of Fig. 3. In other words, during early photomorphogenesis under red light, CCA1/LHY act as negative regulators independently of (or antagonistically to) PRR7/PRR5, which serve as positive regulators. This interpretation is consistent with our previous observation that a CCA1-ox PRR5-ox double transgenic line showed intermediate sensitivity to red light, compared with CCA1-ox (markedly hyposensitive) and PRR5-ox (strongly hypersensitive) (Table 1, Fujimori et al. 2005). Fig. 4 View largeDownload slide Red light responses of a series of homozygous mutants with every possible combination of four mutant alleles (cca1 lhy prr7 prr5). The seedlings (15 types of mutants together with Col) were grown under a given fluence rate of continuous red light (1 μmol m−2 s−1) and in the dark for 3 d. The resulting lengths of their hypocotyls were examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (5 and 10 μmol m−2 s−1), and similar results were obtained (data not shown). For details of construction of these mutants, including a cca1 lhy prr7 prr5 quadruple, see Nakamichi et al. (2007). Fig. 4 View largeDownload slide Red light responses of a series of homozygous mutants with every possible combination of four mutant alleles (cca1 lhy prr7 prr5). The seedlings (15 types of mutants together with Col) were grown under a given fluence rate of continuous red light (1 μmol m−2 s−1) and in the dark for 3 d. The resulting lengths of their hypocotyls were examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (5 and 10 μmol m−2 s−1), and similar results were obtained (data not shown). For details of construction of these mutants, including a cca1 lhy prr7 prr5 quadruple, see Nakamichi et al. (2007). Further genetic analyses employing a set of transgenic lines overexpressing clock-associated genes With regard to the red light signal transduction pathway addressed above, it can be envisaged a priori that the furthest upstream regulators must be the red light photoreceptors. Based on this assumption, combinatorial genetic studies were extended by employing a set of PRR-ox transgenic lines. As shown in Table 1, PRR1-ox and PRR5-ox seedlings display strikingly short hypocotyls at a given fluence rate of red light, while phyA phyB double mutant seedlings are almost insensitive to red light. Based on this rationale, the examined transgenic line here was PRR1-ox phyA phyB. The previously characterized PRR5-ox phyA phyB transgenic line was also employed as an appropriate reference (Matsushika et al. 2007a). These lines were examined with regard to early photomorphogenesis under a given fluence rate of red light (Fig. 5A, 1 μmol m−2 s−1). The effect of PRR5-ox was completely masked in the phyA phyB double mutant background, and the effect of PRR1-ox was also masked considerably, if not completely. The results were confirmed quantitatively (Fig. 5B) (for the PRR5-ox phyA phyB transgenic line, see Matsushika et al. 2007a). The effects of PRR5-ox and PRR1-ox were masked only partially in a phyB single mutant background (data not shown). These results were best interpreted by assuming that the TOC1/PRRs-modulated red light signal transduction is primarily dependent on the phyB photoreceptor, together with phyA. Other members (phyC, phyD and phyE) also appear to be implicated in this event. Fig. 5 View largeDownload slide Red light responses of the PRR5-ox and PRR1-ox transgenic plants in the phyA phyB mutant background with reference to the elongation of hypocotyls. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (1 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) The resulting lengths of their hypocotyls were examined (n > 25). The quantitative result of the PRR5-ox phyA phyB trasngenic line was reported previously (see Matsushika et al. 2007a). Note that mutant plants used in this experiment were all in the Ler background. Note also that we confirmed that the PRR5-ox and PRR1-ox transgenic lines used in this study did indeed produce a large amount of the transcript, as compared with in the case of the wild type (data not shown). Error bars represent SD values. Fig. 5 View largeDownload slide Red light responses of the PRR5-ox and PRR1-ox transgenic plants in the phyA phyB mutant background with reference to the elongation of hypocotyls. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (1 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) The resulting lengths of their hypocotyls were examined (n > 25). The quantitative result of the PRR5-ox phyA phyB trasngenic line was reported previously (see Matsushika et al. 2007a). Note that mutant plants used in this experiment were all in the Ler background. Note also that we confirmed that the PRR5-ox and PRR1-ox transgenic lines used in this study did indeed produce a large amount of the transcript, as compared with in the case of the wild type (data not shown). Error bars represent SD values. Linkage between TOC1 (PRR1) and other PRRs To gain insight into genetic linkage between TOC1 (PRR1) and other PRRs, the following plant lines were also examined: a PRR1-ox prr5 transgenic line and a PRR5-ox toc1 transgenic line (see Table 1). These seedlings were characterized in terms of early photomorphogenesis under a wide range of fluence rates of red light. The results showed that the positive effect of PRR1-ox was fundamentally independent of the existence of the PRR5 gene (Fig. 6). A similar conclusion was obtained from analysis of the PRR5-ox toc1 transgenic line (Table 1, Matsushika et al. 2007a). These results suggested that the functions of TOC1 (PRR1) and PRR5 are integrated into the red light signal transduction pathway independently, at least in part. In this connection, a series of epistasis analyses remain to be carried out with the mutant combinations of toc1, prr7 and prr5, although such a study would be rather difficult because these mutant alleles display the same phenotype of long hypocotyls. Fig. 6 View largeDownload slide Red light responses of the PRR1-ox prr5 double mutant in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (0.05 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) Fluence rate response curves regarding the length of hypocotyls. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of red light. The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 1. Note that we confirmed that the PRR1-ox prr5 transgenic line used in this study did indeed produce a large amount of the PRR1 transcript, as compared with the case of the wild type (data not shown). Fig. 6 View largeDownload slide Red light responses of the PRR1-ox prr5 double mutant in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (0.05 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) Fluence rate response curves regarding the length of hypocotyls. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of red light. The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 1. Note that we confirmed that the PRR1-ox prr5 transgenic line used in this study did indeed produce a large amount of the PRR1 transcript, as compared with the case of the wild type (data not shown). Additional genetic analyses Other genes that should be incorporated into the analyses of this study are PIF3 and PIL6 (see Introduction). To this end, we established the following set of homozygous double mutant lines: phyB pif3, phyB pil6, phyB prr7 and prr7 pif3. Together with each single mutant, they were characterized in the context of this genetic study (Fig. 7A). These results were also examined quantitatively (Fig. 7B). The observed events were best interpreted by assuming that phyB is epistatic to pif3, pil6 and prr7. This is consistent with the view that phyB interacts directly with the downstream PIF3 transcription factor (Duek and Fankhauser 2005). Interestingly, the results also showed that prr7 is epistatic to pif3. This observation is compatible with the idea that PRR7 acts as a positive regulator downstream of PIF3, which serves as a negative effector immediately downstream of phyB in the light signaling pathway in question. Fig. 7 View largeDownload slide Red light responses of additional clock-associated and/or red light signaling-associated mutants in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of mutants was examined. They were grown under a given fluence rate of continuous red light (upper panel) and in the dark (lower panel) for 3 d. Among many seedlings examined (n > 25), two representatives were selected for each. (B) The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 5. Fig. 7 View largeDownload slide Red light responses of additional clock-associated and/or red light signaling-associated mutants in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of mutants was examined. They were grown under a given fluence rate of continuous red light (upper panel) and in the dark (lower panel) for 3 d. Among many seedlings examined (n > 25), two representatives were selected for each. (B) The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 5. In addition to those characterized above, we have been establishing a series of mutant and transgenic lines, which are relevant to this and other circadian clock-associated studies. Here, we summarized the results for these lines in terms of red light sensitivity during early photomorphogenesis (Table 1). Together with the extensive quantitative results presented above, these semi-quantitative results were also taken into consideration in constructing a tentative genetic model, which we finally propose in this study (Fig. 8). Fig. 8 View largeDownload slide A schematic representation of the proposed view regarding the linkages between the circadian-associated components and the phytochrome-dependent red light signal transduction in A. thaliana. Red light is perceived by phytochromes (mainly by phyB together with phyA). PIF3 (perhaps PIL6 too) negatively regulates the phyB-dependent immediate-early signal transduction pathways. This signal transduction pathway is important for the inhibition of hypocotyl elongation in response to a given fluence rate of red light. The clock components: CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (together with PRR9) also play crucial roles close to this red light signal transduction pathway in the manners proposed schematically. In particular, we showed that CCA1/LHY negatively regulates TOC1 (PRR1) that functions as a positive regulator for the red light signaling pathway in question. This genetic interaction between CCA1/LHY and TOC1 is apparently coincident with that seen in the CCA1/LHY–TOC1 negative/positive single-loop clock model (Alabadi et al. 2001) (see a single-loop clock model in the circle). Other details of this tentative model were discussed in the text. Fig. 8 View largeDownload slide A schematic representation of the proposed view regarding the linkages between the circadian-associated components and the phytochrome-dependent red light signal transduction in A. thaliana. Red light is perceived by phytochromes (mainly by phyB together with phyA). PIF3 (perhaps PIL6 too) negatively regulates the phyB-dependent immediate-early signal transduction pathways. This signal transduction pathway is important for the inhibition of hypocotyl elongation in response to a given fluence rate of red light. The clock components: CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (together with PRR9) also play crucial roles close to this red light signal transduction pathway in the manners proposed schematically. In particular, we showed that CCA1/LHY negatively regulates TOC1 (PRR1) that functions as a positive regulator for the red light signaling pathway in question. This genetic interaction between CCA1/LHY and TOC1 is apparently coincident with that seen in the CCA1/LHY–TOC1 negative/positive single-loop clock model (Alabadi et al. 2001) (see a single-loop clock model in the circle). Other details of this tentative model were discussed in the text. Discussion Here, we first interpreted the results of this study by hypothesizing a genetic model (Fig. 8). This proposed model explains the linkage between circadian clock-associated components and phytochrome-dependent red light signal transduction, with special reference to the early photomorphogenesis of seedlings. The model (together with the summarized results in Table 1) provided us with several insights into the signaling mechanism underlying regulation of the length of hypocotyls in response to red light. Briefly, during early photomorphogenesis, a red light signal is perceived by phytochromes. It was previously suggested that phyA dominates at the very early stage (within 1 h) of the red light response (Tepperman et al. 2006). Accordingly, phyA seems to act transiently to inhibit the elongation of hypocotyls. Then, phyB is exclusively responsible for the long-term red light-mediated inhibition of hypocotyl growth (Tepperman et al. 2006). Other phytochromes (phyC, phyD and phyE) might also contribute partly to hypocotyl growth inhibition. PIF3 and PIL6 act immediately downstream of phyB by directly interacting with the photoreceptor, and these bHLH transciption factors negatively regulate the red light signaling pathway, at least with regard to the elongation of hypocotyls. The functions of PIF3 and PIL6 do not appear to be completely redundant because each single null mutant displays an evident phenotype with short hypocotyls under a given fluence rate of red light. In any case, the phyB-dependent signal is integrated into the downstream signaling pathway regulating the length of hypocotyls. Somewhere downstream of the pathway, the clock components, CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (perhaps together with PRR9), play coordinate but distinct roles. Here, CCA1/LHY serve as negative regulators that are partially redundant. The negative effects of CCA1/LHY are dependent on the function of TOC1 (PRR1), which positively regulates the signaling pathway in question. This mode of interaction between CCA1/LHY and TOC1 (PRR1) is coincident with the mode of interaction in the canonical single-loop model for the clock function (see Fig. 8). Interestingly, PRR7/PRR5 also act as partially redundant positive regulators, although PRR7 plays a prominent role. This PRR7/PRR5 positive effect appears to be independent from the branch of CCA1/LHY–TOC1 (PRR1). Through these connections, the clock-associated components have close linkage with the red light signal transduction pathway that regulates the elongation of hypocotyls. Consequently, certain mutations or misexpression of these clock-associated genes result in each characteristic phenotype with regard to red light sensitivity during early photomorphogenesis, as summarized in Table 1. With regard to this model, the toc1-2 single mutant is hyposensitive not only to red light, but also to far-red light (but not to blue light), and the PRR1-ox and PRR5-ox transgenic lines are hypersensitive to both red and far-red light signals (Sato et al. 2002, Mas et al. 2003, Nakamichi et al. 2005b). Together with the observation that CCA1-ox is less sensitive to both red and far-red light in the same assay (Fujimori et al. 2005), it is most likely that this signaling pathway is linked to far-red light-dependent control of hypocotyl elongation. Furthermore, the proposed signaling pathway must overlap with as yet unknown pathways for regulating the opening and expansion of cotyledons in response to red light signals, and also to a pathway for regulating chloroplast development (or greening, see Kato et al. 2007). In this study, however, we will not address these issues in order to keep the model as simple as possible. In addition to CCA1/LHY and PRRs, several other components play roles close to the central clock, including GI, ZTL, LUX (PCL1) and ELF4. Certain mutants of these clock-associated genes also show altered sensitivity to red light: gi, hyposensitive; ztl, hypersensitive; lux, hyposensitive; and elf4, hyposensitive (Huq et al. 2000, Khanna et al. 2003, Somers et al. 2004, Onai and Ishiura 2005). These observations strongly support the view that there are certain linkages between these clock-associated components and red light signal transduction. In fact, the cca1 lhy double mutant is epistatic to gi-3 with regard to red light sensitivity (Mizoguchi et al. 2005). Hence, other clock-associated components should also be integrated into the model to gain a more sophisticated picture. With regard to the proposed model, Quail and colleagues already characterized the molecular basis of the interaction between phyB and PIF3 through biochemical approaches (for a review, see Duek and Fankhauser 2005). The function of PIF3 as a transcription factor appears to be directly regulated by the active (Pfr) form of phyB through a physical interaction (Ni et al. 1999, Al-Sady et al 2006). Aside from this, we do not know anything about the molecular bases of the other mechanisms by which TOC1 (PRR1) and PRR7/PRR5 independently and positively modulate the red light signaling pathway. It is not clear whether TOC1 (PRR1) and PRR7/PRR5 act on the same target in the inferred pathway, although we a priori assume that they do (see Fig. 8, the component denoted by X). In any case, identification of this as yet unknown downstream component must await further examination. We previously suggested that TOC1 (PRR1) has the ability to interact physically with PIF3 (Yamashino et al. 2003). At present, however, this finding is not easily incorporated into the model. In this context, it may also be noted that a pif3 loss-of-function mutant does not compromise clock function (Viczian et al. 2005). It would be of interest to consider the physiological significance of this genetic linkage. The rate of elongation of hypocotyls oscillates diurnally, with a maximum rate at subjective dusk (Dowson-Day and Millar 1999, and for a review, see Nozue and Maloof 2006). This is indicative of the regulation of elongation of hypocotyls by circadian rhythms. Clock-associated red light signal transduction might also be linked to naturally occurring shade avoidance, which is mediated mainly by the ratio of red and far-red light, which is perceived mainly by phyB during plant growth (for a review, see Franklin et al. 2005). Another member of the PIF/PIL family, PIL1, is implicated in a shade avoidance phenomenon in a manner dependent on TOC1 (PRR1) (Salter et al. 2003). Furthermore, one can a priori envisage that not only the light intensity, but also the light quality (or spectrum) fluctuates diurnally and rhythmically, and, thus, plants must anticipate and prepare for such changes in light. Here might be a reasonable basis in plants for the existence of a physiological link between circadian clock function and red light signal transduction. In conclusion, the results of this study allowed us to propose a genetic model regarding a link between the circadian clock and the red light signaling pathway in A. thaliana. Nevertheless, the molecular bases underlying this proposed genetic model remain to be clarified. The critical question of whether these clock-associated components play essentially the same or distinct molecular roles in these apparently disparate processes (i.e. generation of circadian rhythms and modulation of red light signaling) also remains to be answered. In this connection, our model of this study will provide us with the first basis for addressing the long-standing issue as to whether the functions of the clock-associated components are somehow bipartite: on the one hand they function as clock components, and on the other hand they serve as regulators of red light signaling. In this respect, in this study, we showed that CCA1/LHY negatively regulates TOC1 (PRR1), which functions as a positive regulator for the red light signaling pathway in question. Intriguingly, this proposed genetic interaction between CCA1/LHY and TOC1 (PRR1) in the light responses is apparently coincident with that seen in the CCA1/LHY–TOC1 (PRR1) negative and positive single-loop clock model (see Fig. 8). Finally, it is worth mentioning that circadian clock-associated mutants commonly display another hallmark phenotype with regard to the photoperiodic control of flowering time. It is thus of interest to characterize the set of mutants (Table 1) with special reference to the photoperiodic control of flowering. The relevant lines of experiments are under way in our laboratory, and the results will be discussed extensively elsewhere (Nakamichi et al. 2007, Niwa et al. 2007). Materials and Methods Plant materials Arabidopsis thaliana (Columbia accession, designated as Col) was mainly used as the wild type unless otherwise noted. Mutant alleles prr9 (prr9-10), prr7 (prr7-11), prr5 (prr5-11), pif3 (pif3-1), pil6 (pil6-1), and transgenic PRR9-ox (overexpression), PRR7-ox, PRR5-ox, PRR3-ox, PRR1-ox and PIL6-ox lines used in this study have been described previously (see Table 1) (Makino et al. 2002, Matsushika et al. 2002a, Mizoguchi et al. 2002, Sato et al. 2002, Ito et al. 2003, Yamamoto et al. 2003, Fujimori et al. 2004, Murakami et al. 2004, Matsushika et al. 2007a, Matsushika et al. 2007b). The transgenic CCA1-ox line (Wang and Tobin 1998) was a gift from Dr. Elaine M. Tobin (UCLA, Los Angeles, CA, USA). The cca1-1 lhy11 mutant allele (Mizoguchi et al. 2002) was introgressed into Col by back-crossing (four times). Three lines homozygous for cca1-1 lhy11 were selected, and all of these lines showed similar phenotypes (early flowering in short days and short hypocotyl) to those of cca1 lhy double mutants (Mizoguchi et al. 2002). One of lines was named cca1-1 lhy11 (Col) line B and used in this work. The toc1 hypomorphic mutant (toc-1-2) was originally in the C24 background (gift from Dr. Steve A. Kay The Scripps Research Institute, CA, USA). Therefore, the toc1-2 mutant was also introgressed into Col by back-crossing (six times), and toc1-2 homozygous lines in Col were selected. It may be worth mentioning that toc1-2 mutants in the Col and C24 background show essentially the same phenotypes (short period in continuous light, early flowering in short days and long hypocotyls under red light). For phy mutants, the phyA-201 phyB-5 double mutant (Ler background) was used (from Dr. G. C. Whitelam), and the phyB-9 mutant (Col background) was also employed (Reed et al. 1993, Reed et al. 1994). Construction of multiple mutants Multiple mutants were usually made by crossing lines homozygous for each mutation as described below. The cca1-1 lhy11 double mutant was crossed with the toc1-2 (Col background) mutant or the prr7-11 prr5-11 double mutant (Nakamichi et al. 2007). Homozygous derivatives with every possible combination of these alleles were established and analyzed in this study (see Table 1). Transgenic lines, designated as phyA pyhB PRR1-ox, phyA phyB PRR5-ox, PRR1-ox prr5-11 and PRR5-ox toc1-2 (C24 background), were all generated stably through introduction of the corresponding 35S::PRRs transgenes into the phyA phyB, prr5-11 and toc1-2 mutants, respectively, by means of conventional Agrobacterium tumefaciens-mediated transformation procedures. Examination of light response in early photomorphogenesis To examine the light response in early photomorphogenesis of plants, seeds were sown on gellan-gum (0.3%) plates containing MS salts without sucrose. They were then kept at 4°C for 48 h in the dark. Then, seeds were exposed to white light for 3 h in order to enhance germination, followed by incubation at 25°C for 21 h again in the dark. Plants were grown for 2 or 3 d under continuous light at a wide range of fluence rates or in the dark. As the light sources for continuous irradiation, light-emitting diodes were used: for red light, STICK-mR (maximum = 660 nm at 30 μmol m−2 s−1 (TOKYO RIKA, Japan). Acknowledgments Thanks are due for the following: CCA1-ox mutant (Dr. Elaine M. Tobin UCLA, Los Angeles, CA, USA), toc1-2 mutant (Steve A. Kay The Scripps Research Institute, CA, USA) and phyA-201 phyb-5 double mutant (G. C. Whitelam University of Leicester, UK). Thanks are also due to The Salk Institute Genomic Analysis Laboratory (San Diego, CA, USA). This study was supported by Grants-in-Aid for scientific research (to T. M.) from the Ministry of Education, Sports, Culture, Science and Technology of Japan. Also, S.I. was supported by the Japan Society for the Promotion of Science Research Fellowship for Young Scientists. 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Genetic Linkages Between Circadian Clock-Associated Components and Phytochrome-Dependent Red Light Signal Transduction in Arabidopsis thaliana

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Oxford University Press
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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0032-0781
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1471-9053
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10.1093/pcp/pcm063
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17519251
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Abstract

Abstract The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB. Introduction For higher plants, light is one of the most prominent environmental signals and it regulates almost all aspects of developmental stages, as well as physiological states (for a review, see Chen et al. 2004). The thorough understanding of light-mediated signal transduction pathways is one of the major current paradigms of plant biology. A number of studies have been carrried out with reference to the mechanisms underlying such light signal transduction pathways in many plant species. By employing the model higher plant Arabidopsis thaliana, here we focus on an instance of the red light signaling pathway that especially regulates the elongation of hypocotyls during early photomorphogenesis, with special reference to its close linkage with the circadian clock. In A. thaliana, circadian rhythms are extremely relevant to a wide range of biological processes, including diurnal changes in photosynthetic activity and photoperiodic control of flowering time (McClung 2000, Mouradov et al. 2002, Eriksson and Millar 2003, Yanovsky and Kay 2003, Salome and McClung 2004, Nozue and Maloof 2006). The results of recent intensive studies have begun to shed light on the molecular mechanisms underlying the central clock (for recent reviews, see Gardner et al. 2006, Mizuno and Nakamichi 2005, McClung 2006). The current best candidates for such clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its partially redundant homolog LHY (LATE ELONGATED HYPOCOTYL), both of which are single MYB-containing transcription factors (Schaffer et al. 1998, Wang and Tobin 1998, Alabadi et al. 2002, Mizoguchi et al. 2002). In addition, five members of the small PRR (PSEUDO-RESPONSE REGULATOR) family, i.e. PRR1, PRR3, PRR5, PRR7 and PRR9, are believed to be another type of clock-associated component (for recent papers, see Farre et al. 2005, Nakamichi et al. 2005a, Nakamichi et al, 2005b, Salome and McClung 2005). The first described representative of PRRs is TOC1 (TIMING OF CAB EXPRESSION) 1, which is identical to PRR1 (Somers et al. 1998, Strayer et al. 2000) [thus referred to as TOC1 (PRR1) in this text, when necessary for emphasis]. It was proposed that these two types of clock-associated genes [CCA1/LHY and TOC1 (PRR1)] form a negative–positive transcriptional feedback loop that generates fundamental circadian rhythms (Alabadi et al. 2001, see also Fig. 8 later). Subsequently, many other clock-associated genes have been identified and incorporated into the single-loop model, and currently somewhat complicated interlocking multiloop models are favorably envisaged (for reviews, see Mizuno and Nakamichi 2005, Gardner et al. 2006, McClung 2006). Furthermore, a consistent multiloop clock model has recently been built through mathematical simulation (Locke et al. 2006, Zeilinger et al. 2006). During the course of these intensive studies, it was recognized that plants carrying a lesion (i.e. loss of function or misexpression) in a given clock-associated gene commonly display a light response-associated phenotype (Dowson-Day and Millar 1999). Under standard laboratory conditions, the length of hypocotyls of seedlings is finely regulated by a given fluence rate of red light during early photomorphogenesis. This event appears to be closely relevant to an aspect of natural shade avoidance (Nozue and Maloof 2006). In this respect, many circadian-associated mutants and transgenic lines, if not all, commonly show characteristic responses (i.e. hyper- or hyposensitivity) to a wide range of red light fluence rates (Sato et al. 2002, Eriksson et al. 2003, Kaczorowski and Quail 2003, Khanna et al. 2003, Mas et al. 2003, Yamamoto et al. 2003, Fujimori et al. 2004, Fujimori et al. 2005, Mizoguchi et al. 2005). For instance, a cca1 lhy double loss-of-function mutant is hypersensitive to red light, resulting in a phenotype of short hypocotyls, whereas a toc1 hyopomorphic mutant is hyposensitive, resulting in a phenotype of long hypocotyls. In other words, there is a close linkage between the regulation of hypocotyl elongation during early photomorphogenesis and circadian clock-associated components. Nevertheless, little is known about the molecular bases underlying such an intriguing linkage. Here we address this particular issue by employing a large number of Arabidopsis mutants and transgenic lines of a set of clock-associated genes, including CCA1/LHY and PRRs (see Table 1). Furthermore, we also employed certain mutant alleles (including phyA, phyB, pif3 and pil6), which appear to be directly relevant to red light signal transduction per se (see Table 1). Arabidopsis thaliana has five homologous red light photoreceptors, or phytochromes (designated as phyA–phyE) (for reviews, see Casal and Yanovsky 2005, Franklin et al. 2005). Among these red light photoreceptors, phyA and phyB appear to play major roles. Such phyA- and phyB-dependent signal transduction pathways have been characterized intensively at the molecular level (for a review, see Quail 2002). To clarify downstream signaling pathways, many phytochrome-interacting proteins have also been identified. Among such transcription factors, a set of basic helix–loop–helix (bHLH) transcription factors is best characterized, and they have been collectively designated as PIF/PIL (PHYTOCHROME-INTERACTING FACTOR/LIKE) proteins (for a review, see Duek and Fankhauser 2005). Representative is PIF3; this transcription factor was extensively characterized in terms of phytochrome-mediated immediate-early signal transduction (Bauer et al. 2004, Monte et al. 2004). The homologous PIL6 is especially interesting in that its expression is under the control of circadian rhythm (Yamashino et al. 2003). Importantly, both PIF3 and PIL6 are implicated in the early photomorphogenesis of young seedlings under red light (Fujimori et al. 2004). Table 1 List of Arabidopsis mutants and/or transgenic lines characterized in this study Mutants and/or transgenic linesa  Background  Red light sensitivity (degree of IEH)b  References  PRR1-ox  Col  Hyper (+++++)  Makino et al. (2002); Sato et al. (2002)  PRR1-ox prr5-11  Col  Hyper (+++++)  This study  PRR5-ox  Col  Hyper (+++++)  Sato et al. (2002)  PRR5-ox toc1-2  C24  Hyper (++++)  This study  PRR7-ox  Col  Hyper (++++)  Matsushika et al. (2007b)  PRR9-ox  Col  Hyper (++++)  Sato et al. (2002)  cca1-1 lhy11  Col  Hyper (+++)  Mizoguchi et al. (2002)  cca1-1 lhy11 prr7-11  Col  Hyper (++)  Nakamichi et al. (2007)  cca1-1 lhy11 prr5-11  Col  Hyper (++)  Nakamichi et al. (2007)  pif3-1  Col  Hyper (++)  Kim et al. (2003)  pil6-1  Col  Hyper (++)  Fujimori et al. (2004)  lhy11  Col  Hyper (++)  Mizoguchi et al. (2002)  cca1-1  Col  Hyper (++)  Green and Tobin (1999)  lhy11 prr5-1  Col  Hyper (+)  Nakamichi et al. (2007)  cca1-1 lhy11 prr7-11 prr5-11  Col  ±  Nakamichi et al. (2007)  Wild type  Col  ±  Nakamichi et al. (2007)  PRR1-ox phyA-201 phyB-5  Ler  ±  This study  prr5-11 pil3-1  Col  ±  This study  prr5-11 pil6-1  Col  ±  This study  prr9-10  Col  Hypo (−)  Ito et al. (2003)c  cca1-1 prr5-11  Col  Hypo (−)  Nakamichi et al. (2007)  cca1-1 prr7-11  Col  Hypo (−)  Nakamichi et al. (2007)  PRR3-ox  Col  Hypo (− −)  Murakami et al. (2004)  prr5-11  Col  Hypo (− − −)  Yamamoto et al. (2003)d  CCA1-ox PRR5-ox  Col  Hypo (− − −)  Fujimori et al. (2005)  prr9-10 prr5-11  Col  Hypo (− − −)  Nakamichi et al. (2005b)  lhy11 prr7-11  Col  Hypo (− − −)  Nakamichi et al. (2007)  prr7-11  Col  Hypo (− − −)  Yamamoto et al. (2003)e  prr7-11 pil6-1  Col  Hypo (− − −)  This study  prr7-11 pif3-1  Col  Hypo (− − −)  This study  CCA1-ox  Col  Hypo (− − − −)  Wang and Tobin (1998); Matsushika et al. (2002b)  toc1-2  C24 Col  Hypo (− − − −) Hypo (− − − −)  Mas et al. (2003); This studyc  cca1-1 toc1-2  Col  Hypo (− − − −)  This study  lhy11 toc1-2  Col  Hypo (− − − −)  This study  cca1-1 lhy11 toc1-2  Col  Hypo (− − − −)  This study  prr9-10 prr7-11  Col  Hypo (− − − −)  Farre et al. (2005); Salome and McClung (2005); Nakamichi et al. (2005b)  cca1-1 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  lhy11 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2005a)  PIL6-ox  Col  Hypo (− − − − −)  Fujimori et al. (2004)  prr9-10 prr7-11 prr5-11  Col  Hypo (− − − − −)  Nakamichi et al. (2005b)  phyB-9 pil6-1  Col  Hypo (− − − − −)  This study  phyB-9 pif3-1  Col  Hypo (− − − − −)  This study  phyB-9  Col  Hypo (− − − − −)  Reed et al. (1993)  phyB-9 prr7-11  Col  Hypo (− − − − −)  This study  phyB-9 prr7-11 prr5-11  Col  Hypo (− − − − −)  This study  PRR5-ox phyA-201 phyB-5  Ler  Hypo (− − − − −)  This study  phyA-201 phyB-5  Ler  Hypo (− − − − −)  Reed et al. (1994)  Mutants and/or transgenic linesa  Background  Red light sensitivity (degree of IEH)b  References  PRR1-ox  Col  Hyper (+++++)  Makino et al. (2002); Sato et al. (2002)  PRR1-ox prr5-11  Col  Hyper (+++++)  This study  PRR5-ox  Col  Hyper (+++++)  Sato et al. (2002)  PRR5-ox toc1-2  C24  Hyper (++++)  This study  PRR7-ox  Col  Hyper (++++)  Matsushika et al. (2007b)  PRR9-ox  Col  Hyper (++++)  Sato et al. (2002)  cca1-1 lhy11  Col  Hyper (+++)  Mizoguchi et al. (2002)  cca1-1 lhy11 prr7-11  Col  Hyper (++)  Nakamichi et al. (2007)  cca1-1 lhy11 prr5-11  Col  Hyper (++)  Nakamichi et al. (2007)  pif3-1  Col  Hyper (++)  Kim et al. (2003)  pil6-1  Col  Hyper (++)  Fujimori et al. (2004)  lhy11  Col  Hyper (++)  Mizoguchi et al. (2002)  cca1-1  Col  Hyper (++)  Green and Tobin (1999)  lhy11 prr5-1  Col  Hyper (+)  Nakamichi et al. (2007)  cca1-1 lhy11 prr7-11 prr5-11  Col  ±  Nakamichi et al. (2007)  Wild type  Col  ±  Nakamichi et al. (2007)  PRR1-ox phyA-201 phyB-5  Ler  ±  This study  prr5-11 pil3-1  Col  ±  This study  prr5-11 pil6-1  Col  ±  This study  prr9-10  Col  Hypo (−)  Ito et al. (2003)c  cca1-1 prr5-11  Col  Hypo (−)  Nakamichi et al. (2007)  cca1-1 prr7-11  Col  Hypo (−)  Nakamichi et al. (2007)  PRR3-ox  Col  Hypo (− −)  Murakami et al. (2004)  prr5-11  Col  Hypo (− − −)  Yamamoto et al. (2003)d  CCA1-ox PRR5-ox  Col  Hypo (− − −)  Fujimori et al. (2005)  prr9-10 prr5-11  Col  Hypo (− − −)  Nakamichi et al. (2005b)  lhy11 prr7-11  Col  Hypo (− − −)  Nakamichi et al. (2007)  prr7-11  Col  Hypo (− − −)  Yamamoto et al. (2003)e  prr7-11 pil6-1  Col  Hypo (− − −)  This study  prr7-11 pif3-1  Col  Hypo (− − −)  This study  CCA1-ox  Col  Hypo (− − − −)  Wang and Tobin (1998); Matsushika et al. (2002b)  toc1-2  C24 Col  Hypo (− − − −) Hypo (− − − −)  Mas et al. (2003); This studyc  cca1-1 toc1-2  Col  Hypo (− − − −)  This study  lhy11 toc1-2  Col  Hypo (− − − −)  This study  cca1-1 lhy11 toc1-2  Col  Hypo (− − − −)  This study  prr9-10 prr7-11  Col  Hypo (− − − −)  Farre et al. (2005); Salome and McClung (2005); Nakamichi et al. (2005b)  cca1-1 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  lhy11 prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2007)  prr7-11 prr5-11  Col  Hypo (− − − −)  Nakamichi et al. (2005a)  PIL6-ox  Col  Hypo (− − − − −)  Fujimori et al. (2004)  prr9-10 prr7-11 prr5-11  Col  Hypo (− − − − −)  Nakamichi et al. (2005b)  phyB-9 pil6-1  Col  Hypo (− − − − −)  This study  phyB-9 pif3-1  Col  Hypo (− − − − −)  This study  phyB-9  Col  Hypo (− − − − −)  Reed et al. (1993)  phyB-9 prr7-11  Col  Hypo (− − − − −)  This study  phyB-9 prr7-11 prr5-11  Col  Hypo (− − − − −)  This study  PRR5-ox phyA-201 phyB-5  Ler  Hypo (− − − − −)  This study  phyA-201 phyB-5  Ler  Hypo (− − − − −)  Reed et al. (1994)  aFor the original reports with regard to these mutant alleles and/or transgenic lines, see the references cited. In this text, however, note also that they were designated simply without the allele numbers, solely for clarity of the text. bThe sensitivity to red light in terms of IEH (inhibition of elongation of hypocotyls) was compared relative to each other. When seedlings with a certain genetic background showed very short hypocotyls at a given fluence rate of red light, this was scored as ‘hyper-sensitive (+++++)’, whereas when they showed very long hypocotyls, this was scored as ‘hypo-sensitive (− − − − −)’. In this sense, it should be noted that these relative values are semi-quantitative. For the corresponding quantitative data, however, see the results of Figs. 1–7, and the appropriate references. cOther groups have also characterized the same prr9 allele (designated as prr9-1, see Eriksson et al. 2003, Michael et al. 2003). dTwo other prr5 alleles (prr5-1 and prr5-3) were reported, which showed similar phenotypes to that shown here for prr5-11 (Eriksson et al. 2003, Salome and McClung 2005). eMichael et al. (2003) reported the same prr7 allele (as prr7-3). However, Kaczorowski and Quail (2003) reported another prr7 allele in a different background, consistently suggesting a link between PRR7 and the red light signal transduction. fIn this study, the original toc1-2 allele in C24 was transferred into Col by extensive introgression, in order to compare its phenotype consistently with others in the Col background (see Materials and Methods). It may be worth mentioning that the resulting toc1-2 mutant with the Col background showed the expected phenotypes: ‘short-period of free-running rhythms of certain clock-controlled genes’, and ‘markedly early flowering’, as well as ‘hypo-sensitiveness to red light’. It may be also noted that toc1-2 is a strong hypomorphic mutant in that the plants produce about 6% of wild-type transcripts (Strayer et al. 2000), but its short period phenotype is essentially the same as that of a toc1 loss-of function mutant, as far as the phenotype regarding circadian rhythm is concerned (Locke et al. 2006). View Large By employing a number of the clock- and phytochrome-associated mutants mentioned above, here we carried out an extensive series of combinatorial genetic and epistatic analyses, in order to clarify the linkage between the circadian clock and the red light signal transduction pathway. Based on these results, we propose a genetic model. Results Early photomorphogenesis of seedlings under red light in clock-associated mutants Seeds of A. thaliana were germinated and grown on MS gellan-gum plates at a given fluence rate of red light for 2 or 3 d. To examine the sensitivity to red light during early photomorphogenesis, the seedlings were scored with regard to their hypocotyl length in comparison with etiolated seedlings. The seedlings examined in this study were a set of clock-associated and red light signaling-associated mutants or transgenic lines, including a PRR1 (TOC1)-overexpressing (ox) transgenic line (designated PRR1-ox), a toc1 hypomorphic mutant, a PRR5-ox transgenic line, a prr7 prr5 double loss-of-function mutant, a CCA1-ox transgenic line, a cca1 lhy double loss-of-function mutant, a pif3 mutant, a pil6 mutant, a phyA phyB double mutant and others (see Table 1 for details, the allele number of each mutation was omitted for clarity in the text). To interpret the genetic data properly in the unified genetic Columbia (Col) background, the original toc1-2 allele from the C24 background (Strayer et al. 2000) and the cca1-1 lhy11 double mutant alleles from the Ler background (Mizoguchi et al. 2002) were introgressed into the Col background by back-crossing repeatedly (more than four times; see Materials and Methods). The results indicated that some of these mutant seedlings (pif3 mutant, cca1 lhy double mutant, PRR5-ox line and PRR1-ox line) are hypersensitive to red light, and others (CCA1-ox, prr7 prr5 double mutant and toc1 mutant) are hyposensitive compared with the wild-type Col seedlings (see Table 1). These observations are consistent with those reported previously by us and other researchers (Sato et al. 2002, Eriksson et al. 2003, Kaczorowski and Quail 2003, Kim et al. 2003, Mas et al. 2003, Yamamoto et al. 2003, Mizoguchi et al. 2005, Nakamichi et al. 2005a). The results consistently implicated that PIF3 and CCA1/LHY act in the phyA/phyB-mediated light-signal transduction pathway as negative regulators, and PRR7, PRR5 and TOC1 (PRR1) act as positive regulators. Based on these interpretations, the main aim of this study was the clarification of genetic linkage between the circadian clock-associated components and phytochrome-dependent red light signal transduction with special reference to early photomorphogenesis of seedlings. Photomorphogenesis of adult plants was not a concern of this study because such processes are very complicated, and are clearly distinct from the event considered in this study. Combinatorial genetics with regard to CCA1/LHY and TOC1 (PRR1) As mentioned above, toc1 seedlings display a phenotype of hyposensitivity to red light, whereas cca1 lhy double mutants show a phenotype of hypersensitivity (Fig. 1A). Thus, these are ideal mutant alleles for epistasis analysis. Hence, a homozygous cca1 lhy toc1 triple mutant line was established. The sensitivity to red light of seedlings was examined compared with those of appropriate references. Similarly to the toc1 single mutant, the cca1 lhy toc1 triple mutant seedlings showed a phenotype of long hypocotyls under the conditions tested (red light, 0.5 μmol m−2 s−1) (Fig. 1A). This result was confirmed under a wide range of fluence rates of red light (Fig. 1B). Although the results were less evident at a given low fluence rate (0.05 μmol m−2 s−1), it was clearly indicated that the cca1 lhy toc1 triple mutant (2-day-old seedlings) did indeed show a phenotype of long hypocotyls at both 0.5 and 5 μmol m−2 s−1. These observations were confirmed by conducting another independent experiment with 3-day-old seedlings (Fig. 1C). Hence, we concluded that the toc1 lesion is epistatic to the cca1 lhy double mutations. Fig. 1 View largeDownload slide Red light responses of the cca1 lhy toc1 triple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) The indicated set of clock-associated mutants was examined. They were grown under a given fluence rate (0.5 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) on MS plates for 2 d. Among many seedlings examined (n > 25), two representatives were photographed for each. (B, C) Fluence rate response curves regarding the length of hypocotyls. Two independent experiments (biological replicates, experiment 1 and experiment 2) were carried out with essentially the same results. These data are presented here. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of continuous red light for 2 d (Exp. 1) or for 3 d (Exp. 2). Error bars represent SD values. Fig. 1 View largeDownload slide Red light responses of the cca1 lhy toc1 triple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) The indicated set of clock-associated mutants was examined. They were grown under a given fluence rate (0.5 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) on MS plates for 2 d. Among many seedlings examined (n > 25), two representatives were photographed for each. (B, C) Fluence rate response curves regarding the length of hypocotyls. Two independent experiments (biological replicates, experiment 1 and experiment 2) were carried out with essentially the same results. These data are presented here. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of continuous red light for 2 d (Exp. 1) or for 3 d (Exp. 2). Error bars represent SD values. To extend these genetic analyses, we established a series of homozygous mutants with every possible combination with regard to the three mutations (cca1, lhy and toc1). These mutants (seven types) together with Col were examined in terms of red light sensitivity at a given fluence rate (Fig. 2A, B). The results were consistent with the view that the toc1 mutation is fully epistatic to both the cca1 and lhy mutations. The results also suggested that the functions of CCA1 and LHY are partially (but not completely) redundant in this respect. These results are consistent with the assumption that the CCA1/LHY homologous components negatively regulate a red light signal transduction pathway through the function of TOC1 (PRR1), which acts downstream as a positive regulator of the pathway. Fig. 2 View largeDownload slide Red light responses of a set of homozygous mutants with every possible combination of three mutant alleles (cca1 lhy toc1). (A) The seedlings (seven types of mutants together with Col) were grown under a given fluence rate of continuous red light (5 μmol m−2 s−1) and in the dark for 3 d. (B) The resulting lengths of their hypocotyls were quantitatively examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (0.5 and 5 μmol m−2 s−1), and similar results were obtained (data not shown). Fig. 2 View largeDownload slide Red light responses of a set of homozygous mutants with every possible combination of three mutant alleles (cca1 lhy toc1). (A) The seedlings (seven types of mutants together with Col) were grown under a given fluence rate of continuous red light (5 μmol m−2 s−1) and in the dark for 3 d. (B) The resulting lengths of their hypocotyls were quantitatively examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (0.5 and 5 μmol m−2 s−1), and similar results were obtained (data not shown). Combinatorial genetics with regard to CCA1/LHY and PRR7/PRR5 As mentioned above, cca1 lhy double mutant seedlings display a phenotype of short hypocotyls under a given fluence rate of red light, while the prr7 prr5 double mutant shows a phenotype of long hypocotyls under the same conditions (Table 1). Hence, these are also ideal mutant alleles for epistasis analyses. This was done by extensively crossing cca1 lhy and prr7 prr5 double mutants (for details, see Nakamichi et al. 2007). The phenotype of isolated homozygous ccal lhy prr7 prr5 quadruple mutant seedlings was first examined under a given fluence rate of red light (Fig. 3A, 10 μmol m−2 s−1). The quadruple mutant seedlings showed moderate sensitivity to red light, similar to wild-type Col seedlings. These results were confirmed by examining the phenotype under a wide range of fluence rates of red light (Fig. 3B). Within the range of red light fluence rates tested (1 and 10 μmol m−2 s−1), the quadruple mutant seedlings showed a phenotype of moderate hypocotyl length that was similar to that of Col, although this phenotype was less clear at a low fluence rate (0.1 μmol m−2 s−1). These results suggested that both pairs of clock components (CCA1/LHY and PRR7/PRR5) regulate the length of hypocotyls in response to red light independently of (or in parallel with) each other. Importantly, these effects of CCA1/LHY and PRR7/PRR5 are antagonistic. This linkage between CCA1/LHY and PRR7/PRR5 is in sharp contrast to that observed between CCA1/LHY and TOC1 (PRR1) (compare the results of Figs. 1, 3). Fig. 3 View largeDownload slide Red light responses of the cca1 lhy prr7 prr5 quadruple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) For the first experiment (Exp. 1), the indicated set of mutant seedlings, together with the wild-type Col seedlings, was grown under a given fluence rate of continuous red light (10 μmol m−2 s−1) and in the dark for 2 d. The resulting lengths of their hypocotyls were quantitatively examined (n > 25). (B) To ensure reproducibility, the independent experiments were carried out (Exp. 2). In B, fluence rate response curves regarding the length of hypocotyls were examined, with results consistent with those in A. The seedlings were grown under a wide range of fluence rates of red light for 3 d. The resulting lengths of their hypocotyls were examined (n > 25). Error bars represent SD values. Fig. 3 View largeDownload slide Red light responses of the cca1 lhy prr7 prr5 quadruple mutant during early photomorphogenesis with reference to the elongation of hypocotyls. (A) For the first experiment (Exp. 1), the indicated set of mutant seedlings, together with the wild-type Col seedlings, was grown under a given fluence rate of continuous red light (10 μmol m−2 s−1) and in the dark for 2 d. The resulting lengths of their hypocotyls were quantitatively examined (n > 25). (B) To ensure reproducibility, the independent experiments were carried out (Exp. 2). In B, fluence rate response curves regarding the length of hypocotyls were examined, with results consistent with those in A. The seedlings were grown under a wide range of fluence rates of red light for 3 d. The resulting lengths of their hypocotyls were examined (n > 25). Error bars represent SD values. To extend these genetic analyses, we established a series of homozygous mutants with every possible combination with regard to the four mutations (cca1, lhy, prr7 and prr5) (for details, see Nakamichi et al. 2007). These mutants (15 types) together with Col were examined in terms of red light sensitivity at a given fluence rate (Fig. 4). The observed hypocotyl length of these mutants varied gradually from the shortest (the cca1 lhy double mutant) to the longest (the prr7 prr5 double mutant). In these experiments, it was rather difficult to determine statistically the exact degrees of phenotypes amongst these mutants relative to each other. However, it was clear that the cca1 lhy prr7 prr5 quadruple mutant showed a hypocotyl length similar to that of the wild type (Col). The results of these combinatorial epistasis analyses were fully consistent with the results of Fig. 3. In other words, during early photomorphogenesis under red light, CCA1/LHY act as negative regulators independently of (or antagonistically to) PRR7/PRR5, which serve as positive regulators. This interpretation is consistent with our previous observation that a CCA1-ox PRR5-ox double transgenic line showed intermediate sensitivity to red light, compared with CCA1-ox (markedly hyposensitive) and PRR5-ox (strongly hypersensitive) (Table 1, Fujimori et al. 2005). Fig. 4 View largeDownload slide Red light responses of a series of homozygous mutants with every possible combination of four mutant alleles (cca1 lhy prr7 prr5). The seedlings (15 types of mutants together with Col) were grown under a given fluence rate of continuous red light (1 μmol m−2 s−1) and in the dark for 3 d. The resulting lengths of their hypocotyls were examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (5 and 10 μmol m−2 s−1), and similar results were obtained (data not shown). For details of construction of these mutants, including a cca1 lhy prr7 prr5 quadruple, see Nakamichi et al. (2007). Fig. 4 View largeDownload slide Red light responses of a series of homozygous mutants with every possible combination of four mutant alleles (cca1 lhy prr7 prr5). The seedlings (15 types of mutants together with Col) were grown under a given fluence rate of continuous red light (1 μmol m−2 s−1) and in the dark for 3 d. The resulting lengths of their hypocotyls were examined (n > 25), and the relative hypocotyl length (length under red light/length in the dark) was calculated. Two independent experiments were conducted under a wide range of fluence rates of red light (5 and 10 μmol m−2 s−1), and similar results were obtained (data not shown). For details of construction of these mutants, including a cca1 lhy prr7 prr5 quadruple, see Nakamichi et al. (2007). Further genetic analyses employing a set of transgenic lines overexpressing clock-associated genes With regard to the red light signal transduction pathway addressed above, it can be envisaged a priori that the furthest upstream regulators must be the red light photoreceptors. Based on this assumption, combinatorial genetic studies were extended by employing a set of PRR-ox transgenic lines. As shown in Table 1, PRR1-ox and PRR5-ox seedlings display strikingly short hypocotyls at a given fluence rate of red light, while phyA phyB double mutant seedlings are almost insensitive to red light. Based on this rationale, the examined transgenic line here was PRR1-ox phyA phyB. The previously characterized PRR5-ox phyA phyB transgenic line was also employed as an appropriate reference (Matsushika et al. 2007a). These lines were examined with regard to early photomorphogenesis under a given fluence rate of red light (Fig. 5A, 1 μmol m−2 s−1). The effect of PRR5-ox was completely masked in the phyA phyB double mutant background, and the effect of PRR1-ox was also masked considerably, if not completely. The results were confirmed quantitatively (Fig. 5B) (for the PRR5-ox phyA phyB transgenic line, see Matsushika et al. 2007a). The effects of PRR5-ox and PRR1-ox were masked only partially in a phyB single mutant background (data not shown). These results were best interpreted by assuming that the TOC1/PRRs-modulated red light signal transduction is primarily dependent on the phyB photoreceptor, together with phyA. Other members (phyC, phyD and phyE) also appear to be implicated in this event. Fig. 5 View largeDownload slide Red light responses of the PRR5-ox and PRR1-ox transgenic plants in the phyA phyB mutant background with reference to the elongation of hypocotyls. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (1 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) The resulting lengths of their hypocotyls were examined (n > 25). The quantitative result of the PRR5-ox phyA phyB trasngenic line was reported previously (see Matsushika et al. 2007a). Note that mutant plants used in this experiment were all in the Ler background. Note also that we confirmed that the PRR5-ox and PRR1-ox transgenic lines used in this study did indeed produce a large amount of the transcript, as compared with in the case of the wild type (data not shown). Error bars represent SD values. Fig. 5 View largeDownload slide Red light responses of the PRR5-ox and PRR1-ox transgenic plants in the phyA phyB mutant background with reference to the elongation of hypocotyls. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (1 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) The resulting lengths of their hypocotyls were examined (n > 25). The quantitative result of the PRR5-ox phyA phyB trasngenic line was reported previously (see Matsushika et al. 2007a). Note that mutant plants used in this experiment were all in the Ler background. Note also that we confirmed that the PRR5-ox and PRR1-ox transgenic lines used in this study did indeed produce a large amount of the transcript, as compared with in the case of the wild type (data not shown). Error bars represent SD values. Linkage between TOC1 (PRR1) and other PRRs To gain insight into genetic linkage between TOC1 (PRR1) and other PRRs, the following plant lines were also examined: a PRR1-ox prr5 transgenic line and a PRR5-ox toc1 transgenic line (see Table 1). These seedlings were characterized in terms of early photomorphogenesis under a wide range of fluence rates of red light. The results showed that the positive effect of PRR1-ox was fundamentally independent of the existence of the PRR5 gene (Fig. 6). A similar conclusion was obtained from analysis of the PRR5-ox toc1 transgenic line (Table 1, Matsushika et al. 2007a). These results suggested that the functions of TOC1 (PRR1) and PRR5 are integrated into the red light signal transduction pathway independently, at least in part. In this connection, a series of epistasis analyses remain to be carried out with the mutant combinations of toc1, prr7 and prr5, although such a study would be rather difficult because these mutant alleles display the same phenotype of long hypocotyls. Fig. 6 View largeDownload slide Red light responses of the PRR1-ox prr5 double mutant in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (0.05 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) Fluence rate response curves regarding the length of hypocotyls. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of red light. The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 1. Note that we confirmed that the PRR1-ox prr5 transgenic line used in this study did indeed produce a large amount of the PRR1 transcript, as compared with the case of the wild type (data not shown). Fig. 6 View largeDownload slide Red light responses of the PRR1-ox prr5 double mutant in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of seedlings was examined under a given fluence rate (0.05 μmol m−2 s−1) of continuous red light (upper panel) and in the dark (lower panel) for 2 d. (B) Fluence rate response curves regarding the length of hypocotyls. Together with the wild-type Col seedlings, the indicated set of mutant seedlings was grown under a wide range of fluence rates of red light. The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 1. Note that we confirmed that the PRR1-ox prr5 transgenic line used in this study did indeed produce a large amount of the PRR1 transcript, as compared with the case of the wild type (data not shown). Additional genetic analyses Other genes that should be incorporated into the analyses of this study are PIF3 and PIL6 (see Introduction). To this end, we established the following set of homozygous double mutant lines: phyB pif3, phyB pil6, phyB prr7 and prr7 pif3. Together with each single mutant, they were characterized in the context of this genetic study (Fig. 7A). These results were also examined quantitatively (Fig. 7B). The observed events were best interpreted by assuming that phyB is epistatic to pif3, pil6 and prr7. This is consistent with the view that phyB interacts directly with the downstream PIF3 transcription factor (Duek and Fankhauser 2005). Interestingly, the results also showed that prr7 is epistatic to pif3. This observation is compatible with the idea that PRR7 acts as a positive regulator downstream of PIF3, which serves as a negative effector immediately downstream of phyB in the light signaling pathway in question. Fig. 7 View largeDownload slide Red light responses of additional clock-associated and/or red light signaling-associated mutants in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of mutants was examined. They were grown under a given fluence rate of continuous red light (upper panel) and in the dark (lower panel) for 3 d. Among many seedlings examined (n > 25), two representatives were selected for each. (B) The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 5. Fig. 7 View largeDownload slide Red light responses of additional clock-associated and/or red light signaling-associated mutants in early photomorphogenesis. (A) A photographic representation of early photomorphogenesis in red light. The indicated set of mutants was examined. They were grown under a given fluence rate of continuous red light (upper panel) and in the dark (lower panel) for 3 d. Among many seedlings examined (n > 25), two representatives were selected for each. (B) The resulting lengths of their hypocotyls were examined (n > 25). Other details were given in the legend to Fig. 5. In addition to those characterized above, we have been establishing a series of mutant and transgenic lines, which are relevant to this and other circadian clock-associated studies. Here, we summarized the results for these lines in terms of red light sensitivity during early photomorphogenesis (Table 1). Together with the extensive quantitative results presented above, these semi-quantitative results were also taken into consideration in constructing a tentative genetic model, which we finally propose in this study (Fig. 8). Fig. 8 View largeDownload slide A schematic representation of the proposed view regarding the linkages between the circadian-associated components and the phytochrome-dependent red light signal transduction in A. thaliana. Red light is perceived by phytochromes (mainly by phyB together with phyA). PIF3 (perhaps PIL6 too) negatively regulates the phyB-dependent immediate-early signal transduction pathways. This signal transduction pathway is important for the inhibition of hypocotyl elongation in response to a given fluence rate of red light. The clock components: CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (together with PRR9) also play crucial roles close to this red light signal transduction pathway in the manners proposed schematically. In particular, we showed that CCA1/LHY negatively regulates TOC1 (PRR1) that functions as a positive regulator for the red light signaling pathway in question. This genetic interaction between CCA1/LHY and TOC1 is apparently coincident with that seen in the CCA1/LHY–TOC1 negative/positive single-loop clock model (Alabadi et al. 2001) (see a single-loop clock model in the circle). Other details of this tentative model were discussed in the text. Fig. 8 View largeDownload slide A schematic representation of the proposed view regarding the linkages between the circadian-associated components and the phytochrome-dependent red light signal transduction in A. thaliana. Red light is perceived by phytochromes (mainly by phyB together with phyA). PIF3 (perhaps PIL6 too) negatively regulates the phyB-dependent immediate-early signal transduction pathways. This signal transduction pathway is important for the inhibition of hypocotyl elongation in response to a given fluence rate of red light. The clock components: CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (together with PRR9) also play crucial roles close to this red light signal transduction pathway in the manners proposed schematically. In particular, we showed that CCA1/LHY negatively regulates TOC1 (PRR1) that functions as a positive regulator for the red light signaling pathway in question. This genetic interaction between CCA1/LHY and TOC1 is apparently coincident with that seen in the CCA1/LHY–TOC1 negative/positive single-loop clock model (Alabadi et al. 2001) (see a single-loop clock model in the circle). Other details of this tentative model were discussed in the text. Discussion Here, we first interpreted the results of this study by hypothesizing a genetic model (Fig. 8). This proposed model explains the linkage between circadian clock-associated components and phytochrome-dependent red light signal transduction, with special reference to the early photomorphogenesis of seedlings. The model (together with the summarized results in Table 1) provided us with several insights into the signaling mechanism underlying regulation of the length of hypocotyls in response to red light. Briefly, during early photomorphogenesis, a red light signal is perceived by phytochromes. It was previously suggested that phyA dominates at the very early stage (within 1 h) of the red light response (Tepperman et al. 2006). Accordingly, phyA seems to act transiently to inhibit the elongation of hypocotyls. Then, phyB is exclusively responsible for the long-term red light-mediated inhibition of hypocotyl growth (Tepperman et al. 2006). Other phytochromes (phyC, phyD and phyE) might also contribute partly to hypocotyl growth inhibition. PIF3 and PIL6 act immediately downstream of phyB by directly interacting with the photoreceptor, and these bHLH transciption factors negatively regulate the red light signaling pathway, at least with regard to the elongation of hypocotyls. The functions of PIF3 and PIL6 do not appear to be completely redundant because each single null mutant displays an evident phenotype with short hypocotyls under a given fluence rate of red light. In any case, the phyB-dependent signal is integrated into the downstream signaling pathway regulating the length of hypocotyls. Somewhere downstream of the pathway, the clock components, CCA1/LHY, TOC1 (PRR1) and PRR7/PRR5 (perhaps together with PRR9), play coordinate but distinct roles. Here, CCA1/LHY serve as negative regulators that are partially redundant. The negative effects of CCA1/LHY are dependent on the function of TOC1 (PRR1), which positively regulates the signaling pathway in question. This mode of interaction between CCA1/LHY and TOC1 (PRR1) is coincident with the mode of interaction in the canonical single-loop model for the clock function (see Fig. 8). Interestingly, PRR7/PRR5 also act as partially redundant positive regulators, although PRR7 plays a prominent role. This PRR7/PRR5 positive effect appears to be independent from the branch of CCA1/LHY–TOC1 (PRR1). Through these connections, the clock-associated components have close linkage with the red light signal transduction pathway that regulates the elongation of hypocotyls. Consequently, certain mutations or misexpression of these clock-associated genes result in each characteristic phenotype with regard to red light sensitivity during early photomorphogenesis, as summarized in Table 1. With regard to this model, the toc1-2 single mutant is hyposensitive not only to red light, but also to far-red light (but not to blue light), and the PRR1-ox and PRR5-ox transgenic lines are hypersensitive to both red and far-red light signals (Sato et al. 2002, Mas et al. 2003, Nakamichi et al. 2005b). Together with the observation that CCA1-ox is less sensitive to both red and far-red light in the same assay (Fujimori et al. 2005), it is most likely that this signaling pathway is linked to far-red light-dependent control of hypocotyl elongation. Furthermore, the proposed signaling pathway must overlap with as yet unknown pathways for regulating the opening and expansion of cotyledons in response to red light signals, and also to a pathway for regulating chloroplast development (or greening, see Kato et al. 2007). In this study, however, we will not address these issues in order to keep the model as simple as possible. In addition to CCA1/LHY and PRRs, several other components play roles close to the central clock, including GI, ZTL, LUX (PCL1) and ELF4. Certain mutants of these clock-associated genes also show altered sensitivity to red light: gi, hyposensitive; ztl, hypersensitive; lux, hyposensitive; and elf4, hyposensitive (Huq et al. 2000, Khanna et al. 2003, Somers et al. 2004, Onai and Ishiura 2005). These observations strongly support the view that there are certain linkages between these clock-associated components and red light signal transduction. In fact, the cca1 lhy double mutant is epistatic to gi-3 with regard to red light sensitivity (Mizoguchi et al. 2005). Hence, other clock-associated components should also be integrated into the model to gain a more sophisticated picture. With regard to the proposed model, Quail and colleagues already characterized the molecular basis of the interaction between phyB and PIF3 through biochemical approaches (for a review, see Duek and Fankhauser 2005). The function of PIF3 as a transcription factor appears to be directly regulated by the active (Pfr) form of phyB through a physical interaction (Ni et al. 1999, Al-Sady et al 2006). Aside from this, we do not know anything about the molecular bases of the other mechanisms by which TOC1 (PRR1) and PRR7/PRR5 independently and positively modulate the red light signaling pathway. It is not clear whether TOC1 (PRR1) and PRR7/PRR5 act on the same target in the inferred pathway, although we a priori assume that they do (see Fig. 8, the component denoted by X). In any case, identification of this as yet unknown downstream component must await further examination. We previously suggested that TOC1 (PRR1) has the ability to interact physically with PIF3 (Yamashino et al. 2003). At present, however, this finding is not easily incorporated into the model. In this context, it may also be noted that a pif3 loss-of-function mutant does not compromise clock function (Viczian et al. 2005). It would be of interest to consider the physiological significance of this genetic linkage. The rate of elongation of hypocotyls oscillates diurnally, with a maximum rate at subjective dusk (Dowson-Day and Millar 1999, and for a review, see Nozue and Maloof 2006). This is indicative of the regulation of elongation of hypocotyls by circadian rhythms. Clock-associated red light signal transduction might also be linked to naturally occurring shade avoidance, which is mediated mainly by the ratio of red and far-red light, which is perceived mainly by phyB during plant growth (for a review, see Franklin et al. 2005). Another member of the PIF/PIL family, PIL1, is implicated in a shade avoidance phenomenon in a manner dependent on TOC1 (PRR1) (Salter et al. 2003). Furthermore, one can a priori envisage that not only the light intensity, but also the light quality (or spectrum) fluctuates diurnally and rhythmically, and, thus, plants must anticipate and prepare for such changes in light. Here might be a reasonable basis in plants for the existence of a physiological link between circadian clock function and red light signal transduction. In conclusion, the results of this study allowed us to propose a genetic model regarding a link between the circadian clock and the red light signaling pathway in A. thaliana. Nevertheless, the molecular bases underlying this proposed genetic model remain to be clarified. The critical question of whether these clock-associated components play essentially the same or distinct molecular roles in these apparently disparate processes (i.e. generation of circadian rhythms and modulation of red light signaling) also remains to be answered. In this connection, our model of this study will provide us with the first basis for addressing the long-standing issue as to whether the functions of the clock-associated components are somehow bipartite: on the one hand they function as clock components, and on the other hand they serve as regulators of red light signaling. In this respect, in this study, we showed that CCA1/LHY negatively regulates TOC1 (PRR1), which functions as a positive regulator for the red light signaling pathway in question. Intriguingly, this proposed genetic interaction between CCA1/LHY and TOC1 (PRR1) in the light responses is apparently coincident with that seen in the CCA1/LHY–TOC1 (PRR1) negative and positive single-loop clock model (see Fig. 8). Finally, it is worth mentioning that circadian clock-associated mutants commonly display another hallmark phenotype with regard to the photoperiodic control of flowering time. It is thus of interest to characterize the set of mutants (Table 1) with special reference to the photoperiodic control of flowering. The relevant lines of experiments are under way in our laboratory, and the results will be discussed extensively elsewhere (Nakamichi et al. 2007, Niwa et al. 2007). Materials and Methods Plant materials Arabidopsis thaliana (Columbia accession, designated as Col) was mainly used as the wild type unless otherwise noted. Mutant alleles prr9 (prr9-10), prr7 (prr7-11), prr5 (prr5-11), pif3 (pif3-1), pil6 (pil6-1), and transgenic PRR9-ox (overexpression), PRR7-ox, PRR5-ox, PRR3-ox, PRR1-ox and PIL6-ox lines used in this study have been described previously (see Table 1) (Makino et al. 2002, Matsushika et al. 2002a, Mizoguchi et al. 2002, Sato et al. 2002, Ito et al. 2003, Yamamoto et al. 2003, Fujimori et al. 2004, Murakami et al. 2004, Matsushika et al. 2007a, Matsushika et al. 2007b). The transgenic CCA1-ox line (Wang and Tobin 1998) was a gift from Dr. Elaine M. Tobin (UCLA, Los Angeles, CA, USA). The cca1-1 lhy11 mutant allele (Mizoguchi et al. 2002) was introgressed into Col by back-crossing (four times). Three lines homozygous for cca1-1 lhy11 were selected, and all of these lines showed similar phenotypes (early flowering in short days and short hypocotyl) to those of cca1 lhy double mutants (Mizoguchi et al. 2002). One of lines was named cca1-1 lhy11 (Col) line B and used in this work. The toc1 hypomorphic mutant (toc-1-2) was originally in the C24 background (gift from Dr. Steve A. Kay The Scripps Research Institute, CA, USA). Therefore, the toc1-2 mutant was also introgressed into Col by back-crossing (six times), and toc1-2 homozygous lines in Col were selected. It may be worth mentioning that toc1-2 mutants in the Col and C24 background show essentially the same phenotypes (short period in continuous light, early flowering in short days and long hypocotyls under red light). For phy mutants, the phyA-201 phyB-5 double mutant (Ler background) was used (from Dr. G. C. Whitelam), and the phyB-9 mutant (Col background) was also employed (Reed et al. 1993, Reed et al. 1994). Construction of multiple mutants Multiple mutants were usually made by crossing lines homozygous for each mutation as described below. The cca1-1 lhy11 double mutant was crossed with the toc1-2 (Col background) mutant or the prr7-11 prr5-11 double mutant (Nakamichi et al. 2007). Homozygous derivatives with every possible combination of these alleles were established and analyzed in this study (see Table 1). Transgenic lines, designated as phyA pyhB PRR1-ox, phyA phyB PRR5-ox, PRR1-ox prr5-11 and PRR5-ox toc1-2 (C24 background), were all generated stably through introduction of the corresponding 35S::PRRs transgenes into the phyA phyB, prr5-11 and toc1-2 mutants, respectively, by means of conventional Agrobacterium tumefaciens-mediated transformation procedures. Examination of light response in early photomorphogenesis To examine the light response in early photomorphogenesis of plants, seeds were sown on gellan-gum (0.3%) plates containing MS salts without sucrose. They were then kept at 4°C for 48 h in the dark. Then, seeds were exposed to white light for 3 h in order to enhance germination, followed by incubation at 25°C for 21 h again in the dark. Plants were grown for 2 or 3 d under continuous light at a wide range of fluence rates or in the dark. As the light sources for continuous irradiation, light-emitting diodes were used: for red light, STICK-mR (maximum = 660 nm at 30 μmol m−2 s−1 (TOKYO RIKA, Japan). Acknowledgments Thanks are due for the following: CCA1-ox mutant (Dr. Elaine M. Tobin UCLA, Los Angeles, CA, USA), toc1-2 mutant (Steve A. Kay The Scripps Research Institute, CA, USA) and phyA-201 phyb-5 double mutant (G. C. Whitelam University of Leicester, UK). Thanks are also due to The Salk Institute Genomic Analysis Laboratory (San Diego, CA, USA). This study was supported by Grants-in-Aid for scientific research (to T. M.) from the Ministry of Education, Sports, Culture, Science and Technology of Japan. Also, S.I. was supported by the Japan Society for the Promotion of Science Research Fellowship for Young Scientists. 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Journal

Plant and Cell Physiology – Oxford University Press

Published: Jul 1, 2007

Keywords: Arabidopsis thaliana Light signaling Circadian clock Hypocotyl elongation Photomorphogenesis

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