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Keywords:

  • ROTUNDIFOLIA3;
  • CYP90C1;
  • CYP90D1;
  • brassinosteroid;
  • leaf development;
  • Arabidopsis thaliana

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Brassinosteroids (BRs), a group of plant steroid hormones, are essential regulators of plant growth and influence a wide range of vital developmental processes, including germination, cell elongation, vascular element differentiation, photomorphogenesis, senescence, pollen fertility, and stress tolerance (Clouse and Sasse, 1998). Many recent studies have focused on the molecular details of the biosynthesis of BRs in plants and the signaling pathways in BR-related mutants with distinctive pleiotropic phenotypic alterations, including dwarfism, compact rosette leaf structure, and reduced fertility (reviewed in Bishop and Yokota, 2001; Clouse, 2002). All of the genes reported to date, however, are involved in the early reactions of BR biosynthesis, and only a few genes responsible for the late steps in the BR biosynthetic pathways have been characterized, except for the 6-oxidases (Bishop et al., 1999; Shimada et al., 2001). The late steps in biosynthesis are essential for the physiological functions of BRs because only the last two metabolites, castasterone (CS) and brassinolide (BL), are considered biologically active in plants (Sakurai, 1999). Thus, identification and analysis of the genes responsible for the late steps of BR biosynthesis is particularly important, not only for understanding the regulation of BR activity but also for the biotechnological manipulation of BR levels in plants.

As reported previously, leaf length in Arabidopsis thaliana is controlled by a cytochrome P450, ROTUNDIFOLIA3 (ROT3; Kim et al., 1998, 1999; Tsuge et al., 1996). The product of the ROT3 gene, CYP90C1, is homologous to the products of genes that catalyze the early steps of BR biosynthesis pathways (Kim et al., 1998). Previously reported data from microarray analyses suggest that ROT3/CYP90C1 might play a role in the biosynthesis of BRs, as the expression of ROT3/CYP90C1 is suppressed by BRs and is enhanced in mutants with defects in BR biosynthesis (Bancos et al., 2002; Goda et al., 2002; Müssig et al., 2002). This is also the case for genes that encode key enzymes in the early steps of BR biosynthesis (Mathur et al., 1998). Unlike other BR biosynthesis mutants, however, the rot3-1 null allele mutant does not have defects in root or stem lengths (Tsuge et al., 1996), does not suffer reduced fertility, and does not exhibit defects in skotomorphogenesis (Kim et al., 1998). Overexpression of ROT3/CYP90C1 specifically promotes the elongation of leaves (Kim et al., 1999), in contrast to the overexpression of other BR biosynthesis genes which causes elongation of stems (Choe et al., 2001).

In the present study, the ROT3/CYP90C1 gene was determined to be responsible for one of the specific steps in the biosynthesis of bioactive BR. In addition, the gene most closely related to ROT3/CYP90C1, CYP90D1, was isolated; CYP90D1 was determined to be involved in BR biosynthesis and may act to regulate BR biosynthesis in the dark. Furthermore, we provide evidence for a connection between BRs and the light response of plants that is mediated by the two cytochrome P450s, CYP90C1 and CYP90D1.

ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

As reported previously (Kim et al., 1998), the amino acid sequences of the ROT3/CYP90C1 gene show high similarity to enzymes involved in BR biosynthesis. The transcription of genes involved in BR biosynthesis is downregulated by BL, the end product of BR biosynthesis (Mathur et al., 1998; Shimada et al., 2001). Our semiquantitative RT-PCR for steady-state transcript levels of the ROT3/CYP90C1 gene showed that seedlings treated with a high concentration of BL contained lower levels of ROT3/CYP90C1 transcripts than did seedlings treated with a low concentration of BL (Supplementary material). To determine the step of BR biosynthesis in which the ROT3/CYP90C1 gene participated, the intermediates of the BR biosynthesis pathway were fed to wild type and rot3-1 mutant plants, and the endogenous levels of the intermediates produced in wild type and rot3-1 mutant plants were measured. Leaf growth was stimulated when plants were dipped in BR solutions containing 0.05% Triton X-100. However, no clear effects were detected when plants were sprayed with BRs (Kim et al., 1998). Thus, plantlets were incubated with intermediates dissolved in Murashige and Skoog (MS) liquid medium with shaking, as described in Experimental procedures. Surprisingly, only CS and BL were able to rescue the rot3-1 phenotype (Figure 1a), for example, the elongation of leaf petioles (Figure 1b); no other intermediates reversed the effect of the rot3-1 mutation on leaf petiole length. CS is synthesized from typhasterol (TY) or from 6-Deoxocastasterone (6-DeoxoCS), while in plants BL is synthesized from CS (Fujioka and Sakurai, 1997a,b; Fujioka et al., 2002). The measurements of endogenous levels of intermediates in whole seedlings were consistent with the feeding experiments: mutants with either of two null alleles of rot3, that is, rot3-1 or rot3-4, accumulated 6-Deoxotyphasterol (6-DeoxoTY) and TY at higher levels than did wild types (Table 1). Similarly, analyses of intermediates in leaves also revealed slightly higher levels of 6-DeoxoTY and TY in rot3-1 mutants than in wild-type plants (Table 2) and revealed reduced levels of 6-DeoxoCS and CS in rot3-1 mutants relative to wild-type levels (Table 2). Moreover, A-1 plants, which overexpress the ROT3/CYP90C1 gene (Kim et al., 1999), accumulated significantly higher levels of 6-DeoxoCS and CS than did wild types (Table 2). These results strongly suggested that the ROT3/CYP90C1 gene was involved in the conversion of TY to CS and that the conversion of 6-DeoxoTY to 6-DeoxoCS was likely catalyzed by ROT3/CYP90C1, which might act as a C-2α hydroxylase.

image

Figure 1. Feeding experiments of exogenous BR-intermediates on seedlings of Arabidopsis thaliana. Effect of the intermediates of BRs on gross morphology of the rot3-1 null mutant (a) and on the elongation rates of leaf petioles of wild-type (Col) and rot3-1 mutant plants (b). Cathasterone (CT), 6- Deoxocathasterone (6-DeoxoCT), Teasterone (TE), 6-Deoxoteasterone (6-DeoxoTE), 3-Dehydroteasterone (3DT), 3-Dehydro-6-Deoxoteasterone (6-Deoxo3DT), Typhasterol (TY), 6-Deoxotyphasterol (6-DeoxoTY), Castasterone (CS), 6-Deoxocastasterone (6-DeoxoCS), Brassinolide (BL). (c) Effects of intermediates of BRs on gross morphology of rot3-4 cyp90d1 double mutants.

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Table 1.  Brassinosteroid (BR) and sterol profiles in whole seedlings
Sterol and BRWild type (Col-0, gl)rot3-1rot3-4cyp90d1cyp90d1/rot3-4 (double)
  1. Values are given in ng g−1 FW. 24-Methylenecholesterol (24MC), Campesterol (CR), Campestanol (CN), 6-Oxocampestanol (6-OxoCN), Cathasterone (CT), 6- Deoxocathasterone (6-DeoxoCT), Teasterone (TE), 6-Deoxoteasterone (6-DeoxoTE), Typhasterol (TY), 6-Deoxotyphasterol (6-DeoxoTY), Castasterone (CS), 6-Deoxocastasterone (6-DeoxoCS), Brassinolide (BL). –, not analyzed; nd, not detected.

24MC33102580986
CR255002280018400
CN898700602
6-OxoCN32.420.163.8
6-DeoxoCT2.241.783.14
6-DeoxoTE0.130.190.110.050.05
6-DeoxoTY2.313.494.310.150.04
6-DeoxoCS3.261.882.583.900.03
CTndndndndnd
TE0.01nd0.010.01nd
TY0.230.380.460.050.01
CS0.410.310.500.410.10
BLndndndndnd
Table 2.  Brassinosteroid (BR) and sterol profiles in leaf extracts
Sterol and BRWild type (Col)rot3-1 (null mutant)A-CYP90D1; rot3-1 (double homo)aA-CYP90D1/-; rot3-1/rot3-1 (hetero line)a A-1 (ROT3 OE)b
  1. aHomozygote or heterozygote line for A-CYP90D1 with homozygous background for the rot3-1 mutation.

  2. bROT3 overexpression line described previously (Kim et al., 1999).

  3. Values are given in ng g−1 FW. nd, not detected.

24MC43807620258038706600
CR3060045600295004030061700
CN410656547647809
6-OxoCN20.128.651.231.345.3
6-DeoxoCT0.711.051.871.211.91
6-DeoxoTE0.0390.0430.260.0660.12
6-DeoxoTY1.141.460.380.201.66
6-DeoxoCS1.920.790.0340.114.65
CTndndndndnd
TEndndndndnd
TY0.0270.0860.0140.0270.067
CS0.0890.0610.0200.0220.18
BLndndndndnd

CYP90D1 is involved in BR biosynthesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

To understand the role of the ROT3/CYP90C1 gene in the biosynthesis of BRs, furthermore, the CYP90D1 gene (At3g13730) of A. thaliana was isolated. CYP90D1 has the greatest similarity to ROT3/CYP90C1 (47% identity at the whole protein level) among members of the CYP90 family (Figure 2). CYP90D1 was cloned by an RT-PCR screening method with primers designed from the conserved regions of CYP90 family proteins, that is, the proline-rich and the heme-binding domains of the ROT3/CYP90C1 gene (Kim et al., 1998; Figure 2a; see Experimental procedures for details). CYP90D1 was highly similar to CYP90D2 (57% identity at the whole protein level) and CYP90D3 (53% identity at the whole protein level), which has recently been identified in rice (Hong et al., 2003). Three members of the CYP90D subfamily are closely related to the CYP90C1 subfamily, and these two subfamilies along with CYP90C1 may be derived from a common ancestral gene (Figure 2b). Hong et al. (2003) concluded from a feeding experiment with BR biosynthetic intermediates that D2/CYP90D2 might be involved in the catalyzing steps of BR biosynthesis.

image

Figure 2. Alignment of the amino acid sequences of CYP90D1 and other members of CYP90 family (a) and a phylogenetic tree including members of the CYP90 family and other related P450s (b). The arrowhead in (a) indicates the T-DNA insertion site in the cyp90d1 knockout mutant. The phylogenetic tree was generated from deduced amino-acid sequences of CYP90 family proteins from Arabidopsis, CYP90A from pea, CYP85A1 from tomato, and CYP90A3, CYP90B2, CYP90D2, and CYP90D3 from rice. The tree was constructed as described previously (Kim et al., 2002). The conserved region of several P450-specific domains, described previously (Kim et al., 1998), was used to construct the tree. Branch lengths are proportional to the number of amino acid substitutions, and the scale bar indicates 0.1 substitutions. Numbers at nodes indicate bootstrap values.

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Semiquantitative RT-PCR for CYP90D1 transcripts was performed, as for ROT3/CYP90C1, to monitor the steady-state transcript levels with and without BL treatment. Seedlings treated with a high concentration of BL had lower CYP90D1 transcript levels than did seedlings treated with a low concentration of BL (Supplementary material). Similar results were recently reported from microarray analysis and from an expression study of BR-related genes (Goda et al., 2002; Müssig et al., 2002), suggesting that ROT3/CYP90C1 and CYP90D1 could be involved in the biosynthesis of BRs.

To determine whether CYP90D1 was functionally redundant with CYP90C1, as suggested by the high sequence similarity, we introduced the 35S CaMV::CYP90D1 construct into wild-type A. thaliana and the rot3-1 mutant. The transgene did not result in any obvious morphological alterations in the wild-type background (S-CYP90D1 in Figure 3a) and did not reverse the rot3-1 phenotype (Figure 3a). An antisense CYP90D1 construct (A-CYP90D1) also failed to induce any morphological defects (Figure 3a) despite a reduced accumulation of the transcript, as indicated by RT-PCR analysis (data not shown). The cyp90d1 mutant, a knockout mutant of CYP90D1 identified from T-DNA insertion lines (this study; see Experimental procedures for details), also had no obvious morphological defects (Figure 3a). The length of the fifth leaves was 13.0 ± 1.1 mm and 13.2 ± 1.2 mm for Columbia wild type and the cyp90d1 mutant, respectively (number of leaves examined >50). However, interestingly, loss of function of CYP90D1 caused severe dwarfism in combination with a rot3 null-mutant background (Figure 3a). The morphological defects of both the rot3-1 mutant with the A-CYP90D1 transgene and the rot3-4 cyp90d1 double mutant (Figure 3a,b) were very similar to those of known mutants with defects in the early steps of the downstream pathway to BR biosynthesis, such as cpd (Figure 3a), which has a defect in C-23α hydroxylation (Szekeres et al., 1996). Shoot length was severely affected by the dosage of A-CYP90D1 (Table 3). At 45 days after sowing, the average shoot length was 39.0 mm for A-CYP90D1/A-CYP90D1; rot3-1/rot3-1 double homozygous plants, 172.1 mm for rot3-1 single mutants, and 109.3 mm for A-CYP90D1/-; rot3-1/rot3-1 heterozygous plants. Inflorescence lengths of the A-CYP90D1/A-CYP90D1; rot3-1/rot3-1 double-homozygous plants and of the rot3-1/rot3-1; A-CYP90D1/- plants were 23 and 64% that of the rot3-1 single mutant inflorescence length, respectively (Table 3), which is normal compared with wild-type plants (Kim et al., 1998; Tsuge et al., 1996). The shoot elongation measurements clearly showed a synergistic effect of the loss of CYP90D1 function with the rot3-1 mutation. In addition, the treatment with exogenous BL weakly recovered the dwarfism of plants that were double homozygous for A-CYP90D1; rot3-1 (Table 3). A-CYP90D1 might disrupt not only CYP90D1 transcripts specifically but also other closely related CYP90 transcripts by RNA-silencing. However, the double mutant for rot3-1 and the cyp90d1 insertion mutation (Figure 3a) displayed a phenotype very similar to that of the double homozygote for rot3-1 and A-CYP90D1. Thus, the effect of A-CYP90D1 could be attributed to a defect in only the CYP90D1 transcript.

image

Figure 3. Phenotypes of wild-type (wt) plant, rot3 alleles, cyp90d1, double mutants, and transgenic plants. (a) Seedling phenotypes of wt (Col), rot3-1, rot3-4, cyp90d1, cpd-1, sense-CYP90D1 (S-CYP90D1), and antisense-CYP90D1 (A-CYP90D1) plants and double mutants are shown as indicated. Note that S-CYP90D1 does not result in morphological alterations of the rot3-1 phenotype (S-CYP90D1; rot3-1). A-CYP90D1 also has no morphological defects. (b) Morphological comparison of A-CYP90D1; rot3-1 (left) with the rot3-1 mutant (right). Note that the A-CTP90D1; rot3-1 double homozygous plant shows severe dwarfism, but the rot3-1 mutant has normal growth of the inflorescence stem.

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Table 3.  Shoot length of rot3-1 single mutant, A-CYP90D1/rot3-1 heterozygous and homozygous plants
PlantsPrimary shoot ength (mm)aPrimary shoot length (mm)-BL treatedbNo. of plants examinedRatio of BL(+)/BL(−)c
  1. aThe primary shoots of 45-day plants grown at long days (16 h light/8 h dark) were measured.

  2. bThe primary shoots of 45-day plants after treatment with 0.1 μm of brassinolide (BL) for 3 days were measured.

  3. cRatio of shoot elongation of treated versus untreated plants of BL.

  4. Values within parenthesis indicate percentage of shoot length of A-CYP90D1/rot3-1 line versus rot3-1 mutant.

rot3-1/rot3-1172.1 ± 54.0 (100)183.4 ± 58.8 (100)961.06
A-CYP90D1/-(hetero) rot3-1/rot3-1109.3 ± 54.0 (64)118.7 ± 42.1 (65)871.09
A-CYP90D1/A-CYP90D1 rot3-1/rot3-1 (double homo)39.0 ± 22.3 (23)47.7 ± 24.2 (26)1061.36

In the feeding experiments with the rot3-1 cyp90d1 double mutant, all intermediates downstream of Teasterone (TE) in the early C6-oxidation pathway and 6-Deoxocathasterone (6-DeoxoCT) in the late C6-oxidation pathway rescued the phenotypic defects of the double mutant, at least in part (Figure 1c). Moreover, the profiles of the levels of endogenous BR intermediates showed that seedlings and leaves of the double-mutant plants and the A-CYP90D1 transgenic plants with a rot3-1 mutant background accumulated higher levels of 6-Oxocampestanol (6-OxoCN) and 6-DeoxoCT than did those of the wild type (Tables 1 and 2). Most of the intermediates generated from 6-Deoxoteasterone (6-DeoxoTE), that is, 6-DeoxoTY, 6-DeoxoCS, TY, and CS, were present at significantly lower levels in the double mutant than in the wild type (Tables 1 and 2). 6-DeoxoTE accumulated to slightly lower levels in the seedling extracts of the double-mutant plants than in those of the wild type (Table 1), but accumulated to higher levels in the leaves of the A-CYP90D1 transgenic plants with a rot3-1-mutant background than in wild-type leaves (Table 2).

The levels of endogenous BR intermediates in cyp90d1 mutant seedlings were similar to levels of most intermediates in the wild-type seedlings, except for 6-DeoxoTE, 6-DeoxoTY, and TY, which were slightly reduced compared with the levels of the wild type (Table 1). These findings suggest that CYP90D1 could be involved in the late steps of the BR biosynthetic pathway, perhaps the conversion of CT to TE and/or 6-DeoxoCT to 6-DeoxoTE, or TE to 3DT and/or 6-DeoxoTE to 6-Deoxo3DT. A complementation test of the cpd mutation by overexpression of CYP90D1 using the CaMV 35S promoter was performed to determine whether CYP90D1 was involved in either the conversion of 6-DeoxoCT to 6-DeoxoTE of the late C6-oxidation pathway and/or the conversion of CT to TE of the early C6-oxidation pathway, which are catalyzed by CPD/CYP90A1 (Szekeres et al., 1996). In spite of detectable expression of the CYP90D1 transgene, CYP90D1 did not rescue the phenotypes of the cpd mutation (data not shown), suggesting that CYP90D1 could not function as a C-23α hydroxylase. These results indicated that CYP90D1 might be involved, together with other redundant genes, in conversion steps of downstream pathways to BR, such as the conversion of TE to 3DT or 6-DeoxoTE to 6-Deoxo3DT.

Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Unlike the previously reported pattern of CPD expression, that is, strong expression in leaf blades and weak expression in petioles (Goda et al., 2002; Müssig et al., 2002), CYP90D1 was very weakly expressed in leaf blades and moderately expressed in petioles (Figure 4b,d). In particular, CYP90D1 was strongly expressed in leaf vascular tissues (Figure 4j–l). Moreover, the CYP90D1 promoter was more active in petioles in darkness than in the light (Figure 4b,d), while the CPD promoter was expressed ubiquitously in leaves, and darkness had no clear effect on the CPD expression level (Goda et al., 2002). The expression pattern of CYP90D1 was also different from that of ROT3/CYP90C1 (Kim et al., 1999; Figure 4a,c). The ROT3/CYP90C1 promoter was ubiquitously active, independent of light (Figure 4a,c); the CYP90D1 promoter was more active in petioles in darkness than in the light (Figure 4b,d). ROT3/CYP90C1 was expressed in all leaf tissues (Figure 4e–h).

image

Figure 4. Expression of a β-glucuronidase (GUS) reporter gene driven by the promoter of the ROT3/CYP90C1 gene and that of the CYP90D1 gene. (a–d) Expression patterns of GUS under control of the ROT3/CYP90C1 promoter (a, c) and of the CYP90D1 promoter (b, d) in the fifth leaves from 14-day-old seedlings grown under continuous light. In each panel, GUS expression is shown in leaves 0, 3, 6, 12, 24, 36, and 48 h after a shift from continuous light to light (a, b) or darkness (c, d). Bar, 10 mm. (e–l) Transverse sections of leaves of a ROT3 promoter-GUS plant (e–h) and a CYP90D1 promoter-GUS plant (i–l). Light-induced (e, f, i, and j) and dark-induced (g, h, k, and l) patterns of expression of GUS in leaf blades (e, g, i, and k) and petioles (f, h, j, and l) are shown. Bar: 500 μm.

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ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

To understand the differential roles of ROT3/CYP90C1 and CYP90D1 in photomorphogenesis, the growth of hypocotyls of wild type, rot3-1, cyp90d1, and rot3-4 cyp90d1 double-mutant plants under different light conditions was analyzed in mutant strains. The hypocotyl lengths of rot3-1 under various light fluencies or wavelengths were 40–78% of those of the wild type (Figure 5a,b). The hypocotyl length of rot3-1 in darkness was slightly reduced in comparison with that of the wild type (Figure 5a,b). The pattern of hypocotyl elongation did not vary greatly with light intensity, but the hypocotyl length of rot3-1 was affected by red and blue light more than by far-red light of 25 μmol m−2 sec−1 (Figure 5b). These results revealed that the rot3-1 mutant had a defect in hypocotyl elongation that was stimulated by full-spectrum light (Figure 5b), indicating that ROT3/CYP90C1 may positively regulate the elongation of the hypocotyl in response to light signals via the biosynthesis of BRs. Although the cyp90d1 single mutant showed no clear defect in hypocotyl elongation (Figure 5a,b), the rot3-1 cyp90d1 double mutant showed severe defects in hypocotyl elongation under all light and dark conditions (Figure 5a). This may be a result of reduced overall levels of BRs (Table 1), as seen in other BR-deficient dwarf mutants such as cpd and dwf4.

image

Figure 5. Elongation of hypocotyls under different light conditions. (a, b) Elongation of hypocotyls grown under different light sources of 5 μmol m−2 sec−1 intensity (a) and 25 μmol m−2 sec−1 intensity (b) for 5 days on 1/2 MS medium (supplemented with 1% sucrose, 0.8% agar) was measured for wild type, rot3-1, cyp90d1, and rot3-4 cyp90d1 double-mutant plants. Mean ± SD are shown for data from triplicate experiments with approximately 10 seedlings for double mutants and more than 17 seedlings for the other strains. Treatments of seedlings with different light sources were carried out, as reported previously (Tsukaya et al., 2002).

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ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

The results of the present study strongly suggest that two closely related CYP90 genes, ROT3/CYP90C1 and CYP90D1, have different roles in the biosynthesis of BRs. Our data suggest that ROT3/CYP90C1 is involved in the conversion of TY to CS, which has been deemed an activation step in the biosynthesis of BRs. However, the feeding experiments and the analysis of endogenous levels of intermediates strongly suggest that CYP90D1 might participate in the conversion of TE to 3DT or 6-DeoxoTE to 6-Deoxo3DT, and/or 3DT to TY or 6-Deoxo3DT to 6-DeoxoTY.

The present data support the above interpretation, with some minor exceptions. With regard to ROT3/CYP90C1, feeding 6-DeoxoCS to rot3-1 mutant plants did not reverse the mutant phenotype (Figure 1a), perhaps because the conversion of 6-DeoxoCS to CS and BL occurs very slowly, as suggested by Kang et al. (2001) who reported that DDWF1 (CYP92A6) might be involved in the conversion of TY to CS and 6-DeoxoTY to 6-DeoxoCS in peas, although no DDWF1 homolog has been identified in A. thaliana. It is also possible that ROT3/CYP90C1 may be involved only in the conversion of TY to CS of the early C6-oxidation pathway.

The present data also suggest that ROT3/CYP90C1 and another P450(s), which remains to be identified, might play redundant roles in the late step of CS biosynthesis in A. thaliana, because the rot3-1 null allele accumulated some CS and did not show defects in the elongation of inflorescence stems or in skotomorphogenesis (Kim et al., 1998; Tsuge et al., 1996; Figure 3b). For example, ROT3/CYP90C1 appeared to be involved in the same steps as DDWF1 (Kang et al., 2001) in the BR biosynthetic pathway, but no protein analogous to DDWF1 in peas has been identified in A. thaliana. Recently, Kim et al. (2004) reported that an unidentified cytochrome P450(s) catalyzed the conversion from TY to CS, 6-DeoxoTY to 6-DeoxoCS, and from CS to BL based on experiments of microsomal enzyme preparation from cultured cells of Phaseolus vulgaris. Taken together, these data indicate that ROT3/CYP90C1 may be involved in these steps with unidentified redundant gene(s) in A. thaliana.

Regarding CYP90D1, the levels of early metabolites such as 6-DeoxoCT, 6-OxoCN and campestanol (CN) were also slightly depressed in our biochemical profile of cyp90d1. If CYP90D1 were involved in the conversion step of 6-DeoxoCT to 6-DeoxoTE, the levels of 6-DeoxoCT and CN would be expected to increase in the cyp90d1 mutant. Our result could be explained by feedback regulation if the drop in the levels of these metabolites (21–38%) was far less than the decrease in the level of 6-DeoxoTE (approximately 62%) between the cyp90d1 mutant and the wild type (Table 1). Moreover, CN, 6-OxoCN and 6-DeoxoCT accumulated in seedlings of the rot3-1 cyp90d1 double mutants and in the leaves of antisense-CYP90D1 transgenic plants with a rot3-1-mutant background (Tables 1 and 2). Most of the intermediates generated from 6-DeoxoTE, that is, 6-DeoxoTY, 6-DeoxoCS, TY, and CS, were present at significantly lower levels in the double mutant than in the wild type (Tables 1 and 2). These findings suggest that CYP90D1 might be involved in the early steps of the BR biosynthesis pathways, namely, the conversion of CT to TE and/or 6-DeoxoCT to 6-DeoxoTE, or TE to 3DT and/or 6-DeoxoTE to 6-Deoxo3DT. As the 35S-CYP90D1 construct could not complement the cpd mutant, CYP90D1 appears to have no ability to replace the activity of CPD/CYP90A1, which catalyzes CT to TE in the early C6-oxidation pathway and/or 6-DeoxoCT to 6-DeoxoTE in the late C6-oxidation pathway (Szekeres et al., 1996). Thus, we conclude that CYP90D1 might be involved in the steps from TE to 3DT or 6-DeoxoTE to 6-Deoxo3DT. This conclusion is also supported by a recent analysis of the ebisu dwarf2 (d2) rice mutant, which revealed that two orthologs of CYP90D1 from rice, CYP90D2 and CYP90D3, were involved in the steps described above (Hong et al., 2003). The knockout mutant of CYP90D1 (designated cyp90d1) had no obvious phenotypic defects, whereas the d2 mutant in rice showed weak dwarfism (Hong et al., 2003). Thus, the contribution of CYP90D1 to the biosynthesis of BRs might be minor in comparison with the effects of other BR biosynthetic genes. An unknown cytochrome P450(s), which is functionally redundant with CYP90D1, might be involved in the same step.

Although we believe that the above interpretation fits most data obtained from our analyses, alternative interpretations are also possible due to some inexplicable data. For example, our interpretation is based on a model of the biosynthetic pathway of BRs (Fujioka and Sakurai, 1997a,b; Fujioka et al., 2002). If there are unknown bypasses for the biosynthesis of CS, the roles of ROT3/CYP90C1 and CYP90D1 may differ. The broad substrate specificities of certain cytochrome P450s (specifically the multifunctional nature of ROT3/CYP90C1 and CYP90D1) should also be considered. For example, CYP79A1 and CYP71E1, which are involved in the biosynthesis of dhurrin in Sorghum bicolor, were reported to be multifunctional cytochrome P450s that catalyzed multiple steps (reviewed in Winkel, 2004). In this sense, we cannot completely exclude other possibilities.

As discussed above, it is possible that complex regulations including unknown branched pathways are involved in BR biosynthesis. Based on an analysis of DWF4-overexpressing transgenic plants, Choe et al. (2001) revealed that the turnover rate of intermediates in the early C6-oxidation pathway may differ from that of the late C6-oxidation pathway. In addition, intermediates in the early C6-oxidation pathway exhibit greater biological activity on etiolated tissues than those in the late C6-oxidation pathway (Fujioka et al., 1997). Furthermore, our data suggest that ROT3/CYP90C1 and CYP90D1 may be involved in different steps with redundant gene(s). Direct measurement of the enzymatic activities of the CYP90C1 and CYP90D1 proteins expressed in yeast cells, as has been carried out for BR C-6 oxidase (Bishop et al., 1999; Shimada et al., 2001, 2003; Urban et al., 1997), would provide convincing evidence for our interpretations. However, as with the other known CYP90 proteins, the enzymatic activities of CYP90C1 and CYP90D1 have not yet been reported in yeast cells, and we were unable to reconstitute them in the present study. This could be due to a lack of certain co-factors in yeast cells or to a mismatch in the membrane association of CYP90s in yeast cells.

Biosynthesis of BRs and photomorphogenesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Several studies of the involvement of cytochrome P450s in the biosynthesis of BRs (e.g., Müssig et al., 2002) suggest regulation of levels of BRs in different tissues under different environmental conditions. In the present analysis, CYP90D1 was expressed in the leaf petiole but not in the leaf blade (Figure 4b), and the expression level of the CYP90D1 promoter was higher in the elongating leaf petiole in darkness than in the short leaf petiole under light conditions (Figure 4b,d). In addition, the expression level of the CYP90D1 promoter was higher in the vascular tissues in the elongating leaf petioles (Figure 4k,l). These results suggest that the expression of CYP90D1 was regulated temporally and spatially during plant development. Shimada et al. (2003) recently described strong expression of CYP90D (our CYP90D1) in stems, but very weak expression in apical shoots, leaves, fruits, and roots, based on RT-PCR analyses. Such organ-specific expression patterns of CYP90 genes also support the idea of organ-specific regulation of BR biosynthesis and suggest that the examined promoter regions reflect the endogenous expression pattern of these genes in vivo.

The developmental programs and the photo-regulation of leaf petiole and leaf blade elongation are differentially controlled in A. thaliana (Kozuka et al., 2004; Tsukaya et al., 2002). Leaves of A. thaliana exhibit a shade-avoidance syndrome in the dark; specifically, leaf petioles are stimulated to elongate, but leaf blades are suppressed. The expression pattern of CYP90D1 coincides with this organ-specific stimulation of elongation under dark conditions, suggesting that CYP90D1 might be involved in this shade-avoidance syndrome as an adaptive response of leaf growth to dark conditions (reviewed in Tsukaya, 2002). As CYP90D1 probably has a minor function in the biosynthesis of BRs, other unknown functionally redundant genes might be involved in the same step. The differential control of BR biosynthesis by these redundant enzymes is likely to be organ specific and developmentally specific (Bancos et al., 2002). The interaction between light signals and BRs has also been strongly suggested by previous analyses of the DET2 (Li et al., 1996) and BAS1 genes (Neff et al., 1999) in A. thaliana. DET2, which encodes steroid 5α-reductase, is involved in the early steps of BR biosynthesis (Fujioka et al., 1997); BAS1, which encodes a cytochrome P450, CYP72B1, identified as a suppressor of a phytochrome B mutant, is involved in the inactivation of BRs (Neff et al., 1999). Recently, Turk et al. (2003) reported that BAS1/CYP72B1 modulates photomorphogenesis primarily through far-red light and revealed that seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis) in part by rapid modulation of brassinosteroid sensitivity and levels in Arabidopsis. Thus, connections between BRs and the light responses of plants mediated by factors such as DET2, BAS1, and ROT3 have an important role in plant development though modulations of BR levels through both BR biosynthesis pathways and BR inactivation pathways.

In the present study, the rot3-1 mutant was shown to have a defect in the growth of hypocotyls stimulated by different light conditions (Figure 5), whereas the expression level of ROT3/CYP90C1 was not affected by light (Figure 4a,c). ROT3/CYP90C1 may provide an interaction between the light and BR pathways by light-dependent modulation of BR levels, and CYP90D1 may provide modulation of BR levels in the dark. Taken together, coordinated biosynthetic and inactivation mechanisms might be critical for plant adaptation to different environmental conditions and for differential tissue responsiveness at various plant developmental stages.

This study provides evidence that the biosynthesis of active forms of BRs is involved in the integration of light signals with growth control. Detailed analyses of the differential roles of CYP90s would be of great benefit for understanding the basic mechanisms of phytohormone regulation during plant morphogenesis. Moreover, manipulation of the CYP90 genes could be important for the control of the morphology of crop and horticultural plants (reviewed in Feldmann, 2001). Unlike previously identified genes involved in the biosynthesis of BRs, CYP90C1 predominantly influences leaves and floral organs (Kim et al., 1999). The combination of CYP90C1 and CYP90D1 presents new possibilities for the design of horticultural novelties.

Plant materials

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Wild-type A. thaliana Columbia (Col), Wassilewskija (Ws-2), rot3-1 (Col background), rot3-4 (knockout line isolated from T-DNA insertion lines in the present study; Ws-2 background), cyp90d1 (knockout line isolated from T-DNA insertion lines in the present study from the enhancer trap lines of T. Jack's laboratory, Dartmouth College, NH, USA; Col gl1 background), and transgenic plants with sense- and antisense-CYP90D1 constructs driven by the CaMV 35S promoter in Col background were used for the analyses. The cyp90d1 mutant had a mutated CYP90D1 locus with the insertion of T-DNA in the fourth exon (1775 bp downstream of the start codon of the genomic DNA). The ROT3 promoter-β-glucuronidase (GUS) transgenic plant (Kim et al., 1999) and the heterozygotes for the cyp90d1 locus described above were used for the histochemical analyses of GUS activity to monitor the expression patterns of the ROT3/CYP90C1 and CYP90D1 genes, respectively, under various light conditions.

Construction and culture of transgenic A. thaliana

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Plants were grown at 23°C under continuous white fluorescent light (67.4 ± 14.0 μmol m−2 sec−1), as described in Tsuge et al. (1996) and Tsukaya et al. (1991). The partial clone of the CYP90D1 gene was obtained using the PCR method. Primers, designed from conserved regions of CYP90 family proteins, that is, the proline-rich domain and the heme-binding domain of the ROT3/CYP90C1 gene (Kim et al., 1998), were P450-sense (5′-GGAAGCTTAGGCTGGCCGGTGATCGGAGAAACC-3′) and P450-antisense (5′-ACCAGGACATAGCCTTTGCCACCACCAAAGGG-3′). The conditions for direct amplification by RT-PCR with a One-Step RT-PCR Kit (TaKaRa, Tokyo, Japan) were one cycle at 50°C for 30 min and 94°C for 2 min, followed by 35 cycles at 94°C for 30 sec, 42°C for 30 sec, and 72°C for 1 min and 30 sec. The 950-bp amplified product was subcloned and sequenced. A full-length 161J14 cDNA clone (obtained from ABRC, Ohio University, Athens, OH, USA), which had high homology with the partial clone, was sequenced. The coding sequence of CYP90D1 was introduced into the pBI101-hygr vector, and the product was used to transform Agrobacterium. Lines of transgenic plants were established by Agrobacterium-mediated transformation, which was performed by a simplified infiltration method, as described by Kim et al. (1999).

Feeding experiments and analysis of endogenous levels of BRs

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

Intermediates in the biosynthetic pathway of BRs were prepared as described in Fujioka et al. (1997). Ten-day-old seedlings grown on MS medium were transferred to petri dishes containing MS liquid medium supplemented with individual intermediates at 10−7 or 10−8 m with 0.05% Triton X-100 and incubated at 23°C under continuous white fluorescent light (60 μmol m−2 sec−1) with shaking at 100 rpm (Kim et al., 1998). To measure the endogenous BR levels in Arabidopsis plants, shoots from seedlings grown for 4 weeks on soil and leaves from seedlings grown for 25 days on soil under 16 h light/8 h dark conditions (Kim et al., 1998) were harvested and lyophilized. At least 40 g fresh weight of seedlings and leaves, collected independently, were used for the analyses of endogenous sterols and BRs by gas chromatography/mass spectrometry, as described in Fujioka et al. (2002).

Light experiments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

The seeds were sterilized in a solution of NaClO (Tsukaya et al., 1991) and then incubated for 3 days in sterile, distilled water at 4°C in the dark. The seeds were sown in petri dishes on 0.8% (w/v) agar containing MS salt and 1.0% sucrose (w/v) and treated with specific light conditions for 5 days, after which hypocotyl lengths were measured. White light was provided by cool-white fluorescent tubes (FL20SSW/18; Toshiba Co. Ltd, Tokyo, Japan). Red light was provided by light-emitting diodes (LEDs) at a maximum wavelength of 660 nm (181360; EYELA Co. Ltd, Tokyo, Japan). Far-red light was provided by LEDs at a maximum wavelength of 735 nm (181390; EYELA Co. Ltd). Blue light was provided by LEDs at a maximum wavelength of 470 nm (181380; EYELA Co. Ltd). Histological detection of GUS activity was performed using 2-week-old plants that had been grown on MS medium, as described elsewhere (Kim et al., 1999; Tsukaya et al., 1991).

Phylogenetic analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

A phylogenetic tree was generated using the Protein Sequence Parsimony Method (PROTPARS), as described in Kim et al. (2002). Topological robustness was assessed by bootstrap analysis with 100 replicates using simple taxon addition (Felsenstein, 1985). The sequences used for alignments were identified by blast searches of GenBank. The sequences conserved in the CYP90 family and homologs were aligned with the clustalw program (Thompson et al., 1994), and alignments were refined manually using the MacClade program (Sinauer Associates, Inc., Sunderland, MA, USA). The conserved sequences of cytochrome P450 domains, such as the proline-rich-, O2-, steroid-, and heme-binding domains, were included, and several short sequences in the amino-terminal regions that could not be unambiguously aligned were excluded from the analysis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References

The authors thank Dr J. Nam (Dong-A University, Korea) for helpful discussions and Mr H. Choi (Dong-A University, Korea), Dr K.H. Cho, Ms M. Kondo, and Ms Y. Ogawa (NIBB, Japan), and Ms M. Sekimoto and Mr M. Kobayashi (RIKEN, Japan) for their assistance with experiments. The authors also thank ABRC (Ohio University, Athens, OH) for providing the 161J14 cDNA clone (CYP90D1). This research was supported by grants from the Plant Diversity Research Center of the 21st Century Frontier Research Program (PF0330503-00 to G.-T.K.) and from the KOSEF/MOST to the Environmental Biotechnology National Core Research Center (R15-2003-002-01002-0 to G.-T. K.); by a Korea Research Foundation Grant (KRF-2002-015-CS0053 to G.-T.K.); and by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan (no. 14036228 to H. T.), the ‘Ground-based Research Announcement for Space Utilization,’ Japan Space Forum (to H. T.), and the Bio-Design Program, Ministry of Agriculture, Forestry, and Fishes of Japan (to H. T.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. ROT3/CYP90C1 is involved in the conversion of TY to CS, the bioactive BR
  6. CYP90D1 is involved in BR biosynthesis
  7. Expression patterns of CYP90C1 and CYP90D1 differ between light conditions and darkness
  8. ROT3/CYP90C1 positively regulates hypocotyl elongation in response to light signals via the biosynthesis of BRs
  9. Discussion
  10. ROT3/CYP90C1 and CYP90D1 are involved in different steps of the downstream pathway in BR biosynthesis
  11. Biosynthesis of BRs and photomorphogenesis
  12. Experimental procedures
  13. Plant materials
  14. Construction and culture of transgenic A. thaliana
  15. Screening of knockout mutants
  16. RT-PCR analysis
  17. Feeding experiments and analysis of endogenous levels of BRs
  18. Light experiments
  19. Phylogenetic analysis
  20. Acknowledgements
  21. Supplementary Material
  22. References
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