Article Figures & Data
Figures
- Figure 1.
Catabolic pathway of active brassinosteroids. CYP72B1 has been hypothesized to inactivate growth-promoting brassinosteroids such as BL and CS via hydroxylation at carbon 26 (Neff et al., 1999).
- Figure 2.
Biological activity of BL and 26-OHBL in Arabidopsis seedlings. Seedlings were grown for 4 d in the presence of either BL (100 nm) or 26-OHBL (100 nm) in 130 μmol m-2 s-1 white light. Hypocotyl lengths were then normalized to seedlings grown in the absence of hormone and expressed as a percentage change. Each bar represents approximately 100 seedlings from three independent replicates. Error bars represent the se after each seedling was normalized to the average hypocotyl length of the same genotype grown in the absence of hormone.
- Figure 3.
Physiological and genetic analysis of CYP72B1 involvement in BL responsiveness and photomorphogenesis in white light. A, RT-PCR analysis of CYP72B1 transcript abundance in the CYP72B1 overexpressor (cyp72b1-ox8), the wild type (Ws-2), and a null allele (cyp72b1-1). B, Hypocotyl responsiveness to increasing concentrations of exogenous BL of 4-d-old cyp72b1-1 and Ws-2 seedlings in the dark or 115 μmol m-2 s-1 white light. Each point represents approximately 100 seedlings combined from three independent replicates. The Ws-2 and cyp72b1-1 means were not significantly different (P > 0.05) when grown in the dark under all conditions tested. The means were significantly different (P < 0.05) in white light at [BL] of 1 nm or greater. Error bars represent the se. C, Hypocotyl responsiveness of 4-d-old cyp72b1-1, Ws-2, and cyp72b1-ox8 seedlings at nine combinations of varying white-light fluence rate and BL concentration. Each column, representing approximately 100 seedlings combined from three replicates, is significantly different (P < 0.05) from its neighboring column (i.e. comparing column pairs where either light intensity or BL concentration has been changed, but not both at once) and from corresponding columns in the other genotypes.
- Figure 4.
Physiological and genetic analysis of the involvement of CYP72B1 in light responsiveness. Hypocotyl lengths are of 4-d-old cyp72b1-1 and Ws-2 seedlings grown in increasing fluence rates of white (A), blue (B), far-red (C), and red (D) light. Error bars represent the se.
- Figure 5.
Physiological and genetic analysis of the involvement of CYP72B1 in BL responsiveness and photomorphogenesis in varying light conditions. Hypocotyl lengths are of 4-d-old seedlings. Hypocotyl lengths of cyp72b1-1 (A) or cyp72b1-ox8 (B) normalized to Ws-2 hypocotyl lengths under the same conditions and expressed as a percentage change. Seedlings were grown in the dark, 27 μmol m-2 s-1 white, 5 μmol m-2 s-1 red, 3 μmol m-2 s-1 blue, or 30 μmol m-2 s-1 far-red light with or without BL supplementation and without Suc supplementation to the medium. Error bars represent the se after each seedling was normalized to the average wild-type hypocotyl length. n = 36 seedlings per treatment.
- Figure 6.
Organ specificity and light regulation of CYP72B1 transcript accumulation. CYP72B1 (A) and internal reference RNA (B; UBQ10) were accurately and reproducible measured from a 2-fold serial dilution of total cDNA by real time RT-PCR using light upon extension fluorescent primers. RNA abundance is measured as the PCR cycle that generates a level of fluoresce above background or cycle threshold (Ct). Error bars represent the se. C, Whole-seedling CYP72B1 RNA abundance of 4-d-old Col-0 seedlings grown in the dark or shifted to white, red, blue, or far-red light for 1 h and of seedlings with a null allele of PHYA (phyA-211), PHYB (phyB-9), or CRY1 (cry1-102) after a 1-h shift to white light. D, Level of CYP72B1 RNA abundance in the apex, hypocotyl, and root of dark-grown seedlings and seedlings shifted to white light for 1 h. RNA abundance in C and D is reported as the ratio of CYP72B1 to UBQ10 RNA as determined by the Ct and expressed in relative units.
- Figure 7.
Organ specificity and light regulation of CYP72B1 protein accumulation. Photographs of 4-d-old transgenic seedlings containing the GUS gene fused to the N terminus of the CYP72B1 full gene and promoter. The elongation zone of the hypocotyl just below and including the apical hook (A–E) and the root/hypocotyl transition zone (F–J) are pictured. In each light condition, the tissues photographed are from the same seedling. Seedlings were grown in the dark (A and F) and were transferred for 24 h to far-red (B and G), blue (C and H), red (D and I), or white (E and J) light. Seedlings that were not transformed with the GUS reporter fusion did not show GUS staining (not pictured).
Tables
- Table I.
GC-MS analysis Identification of 26-hydroxylated metabolites converted from BL, [2H6]BL, CS, and [2H6]CS in transformed yeast cultures and Arabidopsis seedlings. Yeast were transformed with pED8, which contains the CYP72B1 open reading frame under the control of a Gal-inducible promoter. Arabidopsis seedlings tested were the wild type (Ws-2), a CYP72B1 null (cyp72b1-1), and a CYP72B1 overexpressor (cyp72b1—ox8). All experimental replicates = 1 n.
Yeast Arabidopsis Seedlings Compound Retention Time Characteristic Ions m/z (with Relative Intensities) cyp72b1-1 Ws-2 cyp72b1-ox8 min μg 26-OH brassinosteroid detected 26-OHBL standard 13.72 736 [M+] (5), 721 (7), 619 (17), 577 (14), 564 (13), 299 (25), 156 (100) BL metabolite 13.72 736 [M+] (4), 721 (6), 619 (17), 577 (15), 564 (14), 299 (27), 156 (100) 0.15 1.2 2.2 [2H6] BL metabolite 13.70 742 [M+] (4), 727 (6), 625 (16), 583 (13), 570 (12), 299 (25), 156 (100) 0.08 1.4 2.4 26-OHCS standard 12.25 720 [M+] (3), 705 (24), 603 (100), 514 (27) CS metabolite 12.25 720 [M+] (3), 705 (23), 603 (100), 514 (29) 0.05 0.4 1.7 [2H6] CS metabolite 12.23 726 [M+] (2), 711 (23), 609 (100), 520 (27) 0.01 0.4 1.5
