The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis

Abstract

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]ent-kaurene. The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant. Taken together, our results show that the GA2 locus encodes KS.

Figure 1

Expression of CmKS cDNA in the ga2 mutant. A, The ga2–1 mutant (ga2, an extreme dwarf), the ga2–1 mutant expressing CmKS cDNA (ga2/CmKS), and a wild-type plant (wt). The plants were 35 d old. B, Autoradiogram of an RNA blot showing expression of the CmKS cDNA. Poly(A⁺) RNAs (approximately 3 μg) isolated from wild type (wt), ga2, and the ga2–1 mutant transformed with pBI/KSB101 (lines 7–1-3, 17–1-4, and 18–1-6) were probed with radiolabeled CmKS cDNA and then reprobed with a cyclophilin cDNA (CyP).

Figure 2

Physical map of the AtKS gene. The top bar represents the genomic clone pgAtKS1 with the XbaI restriction map. The RACE products and pAtKS4 (containing the coding region) are cDNA clones. Horizontal thin lines in the cDNA clones show introns and black boxes show exons. Gray bars indicate the stretches of cDNA where a corresponding genomic sequence was not obtained in this study. The position of the first ATG codon and the direction of the open reading frame are indicated by an arrow.

Figure 3

Sequence alignment of plant terpene cyclases. Deduced amino acid sequences of CmKS (Yamaguchi et al., 1996), GA1 (A. thaliana CPS; Sun and Kamiya, 1994), and TEAS from Nicotiana tabacum (Facchini and Chappell, 1992) were compared with that of AtKS (GA2). Amino acids conserved between GA2 and at least one other cyclase are highlighted in reverse print. The amino acids that are proposed to be involved in the formation of the active site of TEAS (Starks et al., 1997) are indicated with asterisks below the sequence. The conserved DDXXD motif is indicated with a line with double arrowheads. The 32-amino acid stretch, which is highly conserved between GA2 and CmKS, is highlighted with a thick horizontal line. The position of the mutation in ga2–1 (Q to a stop codon) is shown with an arrow. Dots are gaps for optimization of alignments.

Figure 4

Functional studies of the AtKS gene from wild-type and the ga2–1 mutant. A, Immunoblot containing total cell lysates from E. coli carrying pET28c (control), pET/AtKS (wt), and pET/ga2 (ga2). The membrane was probed with an alkaline phosphate-conjugated T7-tag antibody. The observed molecular mass of each band is shown on the right. B, KS activities of soluble protein extracts (40 μg of protein each) prepared from E. coli indicated in A. Results are means ± se.

Figure 5

Genomic DNA and RNA blot analyses of the GA2 gene. A, Autoradiogram of a DNA blot containing A. thaliana genomic DNA digested with BamHI (B), EcoRI (E), or HindIII (H). Hybridization was carried out under low-stringency conditions using radiolabeled GA2 cDNA as a probe. B, Autoradiogram of an RNA blot containing poly(A⁺) RNAs purified from 300 μg of total RNAs isolated from different tissues. Aerial tissues (rosette) and roots were harvested from 14-d-old plants grown on agar medium. Cauline leaves, main stems (stem), flower clusters, and young siliques (which contain globular or heart-shaped embryos) were harvested from 35-d-old plants on soil. The membrane was probed with radiolabeled GA2 cDNA and then stripped and reprobed with radiolabeled cyclophilin cDNA (CyP). Numbers below the blot indicate the relative amount of GA2 mRNA standardized by the relative levels of cyclophilin mRNA (“rosette” was arbitrarily set as 1.0).