Selecting genetic transformants of indica and indica-derived rice cultivars using bispyribac sodium and a mutated ALS gene.

Bispyribac sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of acetolactate synthase (ALS) activity. ALS is an enzyme in the biosynthetic pathway for branched-chain amino acids. A mutant form of rice ALS (OsmALS [W548L/S627I]) that confers resistance to BS can be used as an in vitro selection marker gene for plant transformation. Since indica and indica-derived cultivars are thought to have lower BS sensitivity than japonica rice, the application of BS as a selectable reagent for genetic transformation in indica and indica-derived cultivars is more challenging than for japonica cultivars. In this study, callus and seedlings of eight different rice cultivars (five indica-derived cultivars, two indica cultivars and one japonica cultivar) were tested for BS sensitivity. Our study indicates for the first time that callus shows a higher sensitivity to BS than seedlings in indica and indica-derived cultivars. We used BS with OsmALS [W548L/S627I] to select transformed calli, and transgenic rice plants from indica and indica-derived cultivars were successfully obtained.

A rice gene that confers broad-spectrum resistance to ß-triketone herbicides.

The genetic variation of rice cultivars provides a resource for further varietal improvement through breeding. Some rice varieties are sensitive to benzobicyclon (BBC), a ß-triketone herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we identify a rice gene, HIS1 (HPPD INHIBITOR SENSITIVE 1), that confers resistance to BBC and other ß-triketone herbicides. We show that HIS1 encodes an Fe(II)/2-oxoglutarate-dependent oxygenase that detoxifies ß-triketone herbicides by catalyzing their hydroxylation. Genealogy analysis revealed that BBC-sensitive rice variants inherited a dysfunctional his1 allele from an indica rice variety. Forced expression of HIS1 in Arabidopsis conferred resistance not only to BBC but also to four additional ß-triketone herbicides. HIS1 may prove useful for breeding herbicide-resistant crops.

Involvement of EIN3 homologues in basic PR gene expression and flower development in tobacco plants.

The TEIL (Tobacco EIN3-Like) gene is a tobacco homologue of arabidopsis Ethylene Insensitive 3 (EIN3), and the gene product binds an 8 bp sequence in the tobacco PR1a promoter in a sequence specific manner. It was found here that accumulation of TEIL transcript was induced by wounding and preceded basic PR gene expression. To study the downstream signalling pathway of TEIL, TEIL was overexpressed under the control of the constitutive 35S promoter in tobacco plants. In 35S::TEIL lines, basic PR genes, which are wound-, jasmonate-, and ethylene-inducible, were expressed constitutively. Next, the conserved 781 bp sequence among tobacco EIN3-like (EIL) protein genes was introduced as an inverted-repeat (IR) into tobacco to suppress expression of these genes. In two independent IRTEIL lines, the TEIL transcript was not found and transcripts of other tobacco EILs, NtEIL3, and NtEIL5, were reduced. In IRTEIL plants, wound-, jasmonate-, and ACC-induced accumulation of basic PR gene transcripts was significantly inhibited. These results indicate that TEIL functions upstream of tobacco basic PR genes in wound signalling via not only ethylene but also jasmonate. In 35S::TEIL plants, the pistil length of the flower was longer with a slight protrusion of the stigma compared with the control. In IRTEIL plants, the length of the stamens was shorter than the control with significant protrusion of the stigma in the flower. These observations indicate the involvement of tobacco EILs in flower development.

A role of OsGA20ox1 , encoding an isoform of gibberellin 20-oxidase, for regulation of plant stature in rice.

Gibberellin (GA) 20-oxidase (GA20ox) is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. One of the GA20ox genes in rice (Oryza sativa L.), OsGA20ox2 ( SD1 ), is well known as the "Green Revolution gene", and loss-of function mutation in this locus causes semi-dwarfism. Another GA20ox gene, OsGA20ox1, has also been identified, but its contribution to plant stature has remained unclear because no suitable mutants have been available. We isolated a mutant, B142, tagged with a T-DNA containing three CaMV 35S promoters, which showed a tall, GA-overproduction phenotype. The final stature of the B142 mutant reflects internode overgrowth and is approximately twice that of its wild-type parent. This mutant responds to application of both GA3 and a GA biosynthesis inhibitor, indicating that it is a novel tall mutant of rice distinct from GA signaling mutants such as slr1 . The integrated T-DNAs, which contain three CaMV 35S promoters, are located upstream of the OsGA20ox1 open reading frame (ORF) in the B142 mutant genome. Analysis of mRNA and the endogenous GAs reveal that biologically active GA level is increased by up-regulation of the OsGA20ox1 gene in B142. Introduction of OsGA20ox1 cDNA driven by 35S promoter into the wild type phenocopies the morphological characteristics of B142. These results indicate that the elongated phenotype of the B142 mutant is caused by up-regulation of the OsGA20ox1 gene. Moreover, the final stature of rice was reduced by specific suppression of the OsGA20ox1 gene expression. This result indicates that not only OsGA20ox2 but also OsGA20ox1 affects plant stature.