Semidwarf (sd-1), "green revolution" rice, contains a defective gibberellin 20-oxidase gene

Abstract

The introduction of semidwarf rice (Oryza sativa L.) led to record yield increases throughout Asia in the 1960s. The major semidwarfing allele, sd-1, is still extensively used in modern rice cultivars. The phenotype of sd-1 is consistent with dwarfism that results from a deficiency in gibberellin (GA) plant growth hormones. We propose that the semidwarf (sd-1) phenotype is the result of a deficiency of active GAs in the elongating stem arising from a defective 20-oxidase GA biosynthetic enzyme. Sequence data from the rice genome was combined with previous mapping studies to locate a putative GA 20-oxidase gene (Os20ox2) at the predicted map location of sd-1 on chromosome 1. Two independent sd-1 alleles contained alterations within Os20ox2: a deletion of 280 bp within the coding region of Os20ox2 was predicted to encode a nonfunctional protein in an indica type semidwarf (Doongara), whereas a substitution in an amino acid residue (Leu-266) that is highly conserved among dioxygenases could explain loss of function of Os20ox2 in a japonica semidwarf (Calrose76). The quantification of GAs in elongating stems by GC-MS showed that the initial substrate of GA 20-oxidase activity (GA53) accumulated, whereas the content of the major product (GA20) and of bioactive GA1 was lower in semidwarf compared with tall lines. We propose that the Os20ox2 gene corresponds to the sd-1 locus.

Figure 1

Genetic linkage maps and BAC contig of the sd-1 region on chromosome 1 of rice. (A) Genetic linkage map of sd-1 region (16). (B) Physical contig of DNA sequence flanking sd-1 consisting of BAC clones AP003561 and P0408G07 (RGP). (C) High-resolution genetic linkage map of the sd-1 region on chromosome 1 (RGP 2000). Arrow indicates the direction of the centromere.

Figure 2

Alignment of predicted dioxygenase proteins. Dark shading indicates amino acid residues that are conserved in the GA 20-oxidases. GenBank accession numbers are: pea, PsGA20ox, PSU58830; Arabidopsis, AtGA20ox, AAC39314; wheat, TaGA20ox, TAY14007; rice, OsGA20ox1, U50333; OsGA20ox2, AY114310; OsGA3ox, AB056519; AtGA2ox, AJ132435. Arrow indicates the highly conserved leucine residue.

Figure 3

Nucleotide and predicted amino acid sequence of GA 20-oxidase sequence isolated from chromosome 1 of rice. The positions located between 557–558 and between 879–880 are indicated. Arrows denote the position of the 280-bp deletion occurring within the coding sequence in Doongara (sd-1). The last nucleotide (cytosine) before the deletion start (position 297) in Kyeema is changed to an adenine residue in Doongara, resulting in a synonymous nucleotide substitution (Ser TCC → Ser TCA). The conserved leucine Leu-266 that was changed to phenylalanine in semidwarf mutant Calrose76 is highlighted.

Figure 4

Hybridization of DNA probe of exon 2 derived from the putative GA 20-oxidase gene (Os20ox2) to (HindIII) digested genomic DNA of Nipponbare (N, tall), Kyeema (K, tall), Doongara (D, sd-1), IR36 (IR, sd-1), Calrose (C, tall), and Calrose76 (C76, sd-1 allele).

Figure 5

The early 13-hydroxylation pathway of gibberellin biosynthesis in higher plants. Black circles indicate oxidation steps catalyzed by GA 20-oxidase (adapted from ref. 20).