Similarities and Differences between the Characteristics of Gibberellin-Binding Protein and Gibberellin 2-Oxidases in Adzuki Bean (Vigna angularis) Seedlings

Abstract

Gibberellin-binding proteins (GBPs) were purified ca. 230,000 fold. The characteristics of adzuki GBP were examined and compared with those of a recombinant gibberellin 2-oxidase (rVaGA2oxA1) that was fused with glutathione S-transferase (GST). VaGA2oxA1 was most abundantly expressed in etiolated adzuki bean seedlings, and VaGA2oxA1 and GBPs from adzuki bean seedlings showed gibberellin-binding activity when incubated with 2-oxoglutarate and Co 2þ . Both rVaGA2oxA1 and partially purified GBPs from adzuki bean seedlings showed very similar selectivity to gibberellins in binding assays, where biologically active gibberellins such as GA 4 , GA 3 , GA 7 , and GA 1 showed higher binding affinity than biologically inactive gibberellins such as GA 8 , GA 34 , and 3-epi-GA 4 . The polyclonal antibody raised against rVaGA2oxA1 cross-reacted with all rVaGA2oxs (rVaGA2oxA1, rVaGA2oxA2, rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3) whose cDNAs were cloned from adzuki bean seedlings. Treated with the antibody, the recombinants that originally showed gibberellin-binding activity lost both binding activity and enzymatic activity. In contrast to the recombinants, the gibberellin-binding activity of GBPs from adzuki bean seedlings was hardly affected by the antibody treatment. The GBPs showed very weak gibberellin 2-oxidase-like activity, and it was not affected by the antibody treatment either. These observations suggest that a major component that showed GA-binding activity was apparently different from any gibberellin 2-oxidase cloned from the seedlings.

Gibberellin Receptor and Its Role in Gibberellin Signaling in Plants

“…Nakajima et al (43) also detected GA-binding activity in the soluble protein fraction from adzuki bean (Vigna angularis) seedlings by using a gel-permeation column. Further studies have revealed that this GBP in the partially purified fraction fulfills all four of the GA receptor criteria (47,48).…”

Identification and characterization of Arabidopsis gibberellin receptors

“…(1991) and Gilroy and Jones (1994) have shown that a GA receptor is localized on the plasma membrane of barley aleurone cells, while Nakajima et al. (1997) and Park et al. (2005) suggested that GA‐receptor‐like GA‐binding proteins exist in the cytosolic fraction of adzuki bean seedlings.…”

“…Attempts to identify the GA receptor have been made using biochemical and genetic approaches. Hooley et al (1991) and Gilroy and Jones (1994) have shown that a GA receptor is localized on the plasma membrane of barley aleurone cells, while Nakajima et al (1997) and Park et al (2005) suggested that GA-receptor-like GA-binding proteins exist in the cytosolic fraction of adzuki bean seedlings. The genetic approach has enabled many mutants with dwarf phenotypes to be isolated and analyzed to identify the genes responsible for the phenotypes.…”

Trails to the gibberellin receptor, GIBBERELLIN INSENSITIVE DWARF1

“…They partially purified GBPs and analyzed the biochemical properties of the proteins. [46][47][48] The bound [ 3 H] 4 -dihydroGA 4 was replaced by nonlabeled GA 4 , suggesting the reversibility of the binding. While it shows a high affinity specific for so called active GAs such as GA 1 , GA 3 , GA 4 , and GA 7 , the binding affinity for GA 9 , GA 8 , and GA 13 is fairly low.…”

“…They extensively purified the GBP up to 230,000-fold (Table 2) and examined its biochemical property. 47,48) There remains the possibility that GA 2-oxidase (GA2ox) which selectively catalyzes 2-oxidation of active GAs such as GA 4 and GA 1 shows similar property. They cloned five cDNAs (VaGA2oxA1, A2, VaGA2oxB1, B2 and B3) expressed in adzuki bean seedlings, prepared recombinant VaGA2oxs and examined the enzyme activity and the GA-binding activity (Fig.…”