Article Figures & Data
Figures
- Fig. 1.
Two major pathways for ABA metabolism. The bold arrows indicate the major pathways that have been reported to function in plants. The normal arrows indicate the minor pathways (Zeevaart and Creelman, 1988; Zeevaart, 1999; Barthe et al., 2000).
- Fig. 2.
Autoradiographic profile of the [3H]ABA metabolites produced in adzuki. Lane 1 is free [3H]ABA. Lanes 2 to 6 are the extracts from leaves, and lanes 7 to 11, from hypocotyls. Plants were treated with [3H]ABA for 2 h (lanes 2 and 7), 4 h (lanes 3 and 8), 6 h (lanes 4 and 9), and 8 h (lanes 5 and 10). Lanes 6 and 11 are for the extracts from plants pretreated with 5 × 10−5 m non-labeled ABA for 6 h and then incubated in a [3H]ABA solution for 2 h.
- Fig. 3.
cDNA and deduced amino acid sequences of ABA-GTase from adzuki bean seedlings. The broken line indicates the second peroxisomal targeting signal. The boxes indicate theN-glycosylation motif with a pattern N[∧P][ST][∧P]. The underlining shows the UDP-glycosyltransferase signature. The shaded area is the coiled-coil region detected by Lupas's algorithm.
- Fig. 4.
Results of enzyme assays using recombinant protein. A, SDS-PAGE pattern of the recombinant protein. Lane 1 is crude protein prepared from the cell lysate after inducing rAOG by 0.2 mm IPTG for 4 h at 22°C. Lane 2 shows the fusion protein purified by affinity chromatography. B, Reaction products from ABA and UDPG or G-1-P by rAOG. Lanes 1 and 2 and lanes 3 and 4, respectively, indicate the patterns from GST and rAOG. + and −, The presence and absence, respectively, of UDPG or G-1-P in a reaction mixture. C, The pH dependence of ABA-GTase activity expressed inE. coli. The arrowhead indicates the product. D, The signal intensities of the products under different reaction conditions. Reactions were performed without ABA (lane 1), UDPG (lane 2), or rAOG (lane 3), respectively, and with rAOG denatured by boiling for 10 min (lane 4). Lane 5 shows the product using the native rAOG as a positive control. + and −, The presence and absence, respectively, of ABA, UDPG, or rAOG in a reaction mixture. ●, The [3H]ABA. ○, The boiled rAOG.
- Fig. 5.
Identification of the substrate for recombinant GTase. A, Confirmation of the substrate. Lane 1 is free [3H]ABA. Lane 2 is the product from UDPG and [3H]ABA. Lane 3 shows the reaction product from ABA and [14C]UDPG. The reaction mixture was incubated for 2 h at 30°C. B, Reactivity of ABA and the ABA-Me derivative. The mixture was incubated for 4 h at 30°C. F, ABA; M, ABA-Me. The arrowhead indicates the products.
- Fig. 6.
Identification of the product. A, Comparison of RF values between the ABA-GE standard and the product. S, P, and S+P indicate the ABA-GE standard, the product, and the equimolar mixture of the ABA-GE standard and the product, respectively. An arrowhead indicates the position of ABA-GE separated in a solvent of CHCl3:MeOH:AcOH:H2O (40:15:3:2, v/v). B, Pattern for standard ABA-GE and the product treated with 0.2 m NaOMe, having been separated in a solvent of EtOAc:CHCl3:AcOH (25:15:1, v/v). F and M are free ABA and ABA-Me, respectively. R1 and R2, respectively, show the solvolysis products from the enzyme and standard ABA-GE. The upper arrowhead indicates the position of ABA-Me. The lower arrowhead shows the position of free ABA.
- Fig. 7.
Substrate specificity of ABA-GTase. Lanes 1 to 8 show the products of prAOG incubated with [14C]UDPG and salicylic acid, trans-cinnamic acid, trans-ABA, cis-ABA, GA3, IAA, JA, and zeatin, respectively. The upper and lower arrowheads indicate the putative trans-cinnamic acid-glucosyl ester and ABA-GE, respectively.
- Fig. 8.
Characterization of ABA-GTase specificity. Lanes 1 to 4 show the products of prAOG incubated with (+)-S-ABA, (−)-R-ABA, (−)-PA, and 2-trans-ABA, respectively, in the presence of [14C]UDPG. The arrowhead indicates the products discussed in the text.
- Fig. 9.
Northern-blot analysis of AOG mRNA. A, mRNA level expressed in the leaves (L) and hypocotyls (H) with no treatment (lanes 1 and 2), and after being treated with 50 μm ABA for 24 h (lanes 3 and 4), drought for 6 h (lanes 5 and 6), and wounding for 6 h (lanes 7 and 8). B, mRNA levels expressed in the hypocotyls of plants treated with DW (lane 1), GA3 (lane 2), IAA (lane 3), zeatin (lane 4), JA (lane 5), and ABA (lane 6). C, mRNA levels expressed in the hypocotyls of plants subjected to drought for up to 8.5 h. D, mRNA levels expressed in the hypocotyls of plants subjected to wounding for 0.0 to 8.0 h. The upper or lower arrowheads in A through D indicate the AOG transcript and rRNA as an indicator of the total RNA quantity (7 μg/lane), respectively.
- Fig. 10.
Southern-blot analysis of genomic DNA digested with SphI (lane 1), XbaI (lane 2),EcoRI (lane 3), and SalI (lane 4). The amount of DNA loaded was 20 μg/lane. Hybridization and washing were carried out at 65°C and 68°C, respectively.
