Article Figures & Data
Figures
- Figure 1.
Sequence Alignment of GA 3β-Hydroxylases.
An alignment is shown for the deduced amino acid sequences of Arabidopsis GA4 (Chiang et al., 1995, 1997) and GA4H (GenBank accession number AF070937), pea LE (Lester et al., 1997; Martin et al., 1997), and GA 2β,3β-hydroxylase from pumpkin (Cm; Lange et al., 1997). Identical amino acid residues conserved between GA4H and at least one other protein are indicated in reverse type, and similar residues are in gray boxes. Gaps introduced to optimize the alignment are indicated as dots, and sequence truncations are indicated by dashes. The boxes were drawn using the BOXSHADE web site (http://ulrec3.unil.ch/software/BOX_form.html).
- Figure 2.
In Vitro Functional Analysis of the Recombinant GA4H Protein.
(A) Diagram showing conversion of GA9 and GA20 to GA4 and GA1 by 3β-hydroxylation.
(B) Functional analysis of the GA4H protein. Lysates of E. coli containing the MBP (as a control) or MBP–GA4H were incubated with 14C-GA9. The reaction mixture was separated on a silica gel by thin-layer chromatography. The positions of authentic GA4 and GA9 are indicated by arrowheads.
- Figure 3.
Developmental Regulation of GA4 and GA4H Expression.
Autoradiography of RNA gel blots containing 25 μg of total RNAs isolated from different tissues, as labeled. Germinating seeds under continuous white light were harvested 12 hr (h) after imbibition. Young seedlings (harvested at 48, 72, and 96 hr after imbibition) and 14- and 35-day (d)-old plants were grown under 16-hr-light and 8-hr-dark cycles. Aerial tissues (rosette) were harvested from 14-day-old plants. Cauline leaves (leaf), main stems (stem), flower clusters (flower), and siliques (siliques 1, 2, and 3) were harvested from 35-day-old plants. Silique 1 has embryos at globular and heart-shaped stages. Embryos at torpedo to upturn U stages are contained in silique 2. Silique 3 has mature green seeds. The membrane was hybridized with the radiolabeled GA4 or GA4H antisense RNA probe and then reprobed with the the radiolabeled 18S rDNA probe as a loading control.
- Figure 4.
R Light–Induced Expression of the GA4 and GA4H Genes and Germination in Wild-Type Arabidopsis Seeds.
(A) Diagram showing different light treatments. The seeds were imbibed in the dark and then irradiated with an FR light pulse 1 hr after imbibition. The seeds were then either irradiated with an R light pulse (stippled box) 24 hr after the FR light pulse (R in [B]) or incubated without the R light pulse in the dark (D in [B]). The triangle indicates the starting time of imbibition. The vertical arrow indicates the beginning of the R light pulse, which was set as 0 hr in the experiments shown in (B).
(B) Autoradiography of RNA blots containing 12.5 μg of total RNAs prepared from germinating wild-type seeds under different light conditions as described in (A). The time after the R light pulse is indicated above the blot. The membrane was hybridized with the GA4 or GA4H antisense RNA probe and then reprobed with the radiolabeled 18S rDNA probe as a loading control.
(C) Germination frequency and the levels of GA4 and GA4H mRNAs after the R light pulse. The highest mRNA levels for each gene were set as 100.
- Figure 5.
R Light–Induced Expression of the GA4 and GA4H Genes in Imbibed ga1-3 Seeds.
(A) Autoradiography of RNA blots containing 12.5 μg of total RNAs from imbibed ga1-3 seeds after light treatments, as diagrammed in Figure 4A. The membranes were hybridized with the GA4 or GA4H antisense RNA probe and then reprobed with the radiolabeled 18S rDNA probe as a loading control. Abbreviations are as given in Figure 4A.
(B) A diagram showing relative levels of cross-hybridization of the GA4H probe with the GA4 mRNAs (black bars) and the net GA4H mRNA (open bars) in each lane in (A). The highest level of the hybridization signal was set as 100.
(C) GA4 and GA4H mRNA levels after the R light pulse. The highest mRNA levels for each gene were set as 100.
- Figure 6.
Effects of the phyB-1 Mutation on GA4 and GA4H Expression.
(A) Diagram showing different light treatments. The seeds were imbibed in the dark and then irradiated with an FR light pulse 1 hr after the start of imbibition. The seeds were then either irradiated with an R light pulse 2 hr after the FR light pulse (R in [B]) or incubated without an R light pulse in the dark (D in [B]). Symbols are as given in the legend to Figure 4A.
(B) Autoradiography of RNA gel blots containing 12.5 μg of total RNAs prepared from germinating wild-type (WT) or phyB-1 seeds under the light conditions given in (A). The membrane was hybridized with the GA4 or GA4H antisense RNA probe and then reprobed with a radiolabeled 18S rDNA probe as a loading control.
- Figure 7.
Photoreversibility of GA4 and GA4H Expression.
(A) Diagram showing different light treatments. The seeds were imbibed in the dark and then irradiated with an FR light pulse 1 hr after imbibition. The seeds were then incubated in the dark (D), irradiated with an R light pulse (R), or treated with a second FR light pulse immediately after the R light pulse (R+FR). Symbols are as given in the legend to Figure 4A.
(B) Autoradiography of RNA blots containing 12.5 μg of total RNAs prepared from germinating wild-type seeds under the light conditions given in (A). The membrane was hybridized with the GA4 or GA4H antisense RNA probe and then reprobed with a radiolabeled 18S rDNA probe as a loading control.
- Figure 8.
Effects of the ga1-3 Mutant Background and GA4 Application on GA4 and GA4H Expression in Imbibed Seeds.
Shown is autoradiography of RNA gel blots containing 12.5 μg of total RNA from imbibed wild-type (WT) or ga1-3 seeds.
(A) RNA was isolated from seeds at 4 hr after the light treatments, as shown in Figure 4A.
(B) The ga1-3 seeds were imbibed in water (−GA4) or in 100 μM GA4 under continuous white light. Germination percentage is shown below the blots.
The membranes were hybridized with the GA4 or GA4H antisense RNA probe and then reprobed with a radiolabeled 18S rDNA probe as a loading control.
- Figure 9.
Proposed Model for the Regulation of GA4 and GA4H Expression.
The last two steps of the GA biosynthetic pathway are shown in the dashed box. Arrows indicate positive regulation. The feedback inhibition is shown by the T-bar.
