The origin and distribution of 'Kokubu'-type splice-site mutations of the MLO genes in tobacco varieties.

The Japanese domestic tobacco (Nicotiana tabacum L.) cultivar 'Kokubu' shows high powdery mildew resistance that is controlled by splice-site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. We investigated the existence of the same NtMLO1/2 splice mutations in the genomes of various tobacco varieties cultivated in Japan and other countries. In total, 14 Japanese domestic cultivars, which were mainly distributed in Kagoshima, had splice-site mutations in both NtMLO1 and NtMLO2. In addition, tobacco cultivars containing only the NtMLO1 splice-site mutation were found in various tobacco production areas in Japan, but no cultivars with only the NtMLO2 splice-site mutation were detected. Moreover, the NtMLO1 splice-site mutation was detected in native Asian, Oriental and cigar tobacco varieties. Consequently, we speculate that these powdery mildew-resistant tobacco cultivars were generated relative recently in the Kagoshima area when a spontaneous mutation occurred at the NtMLO2 splice site in a cultivar already containing the NtMLO1 splice-site mutation and that the NtMLO1 splice-site mutation occurred during the early period of tobacco seed dissemination from the Americas to Asia and Japan.

Inhibition of lateral shoot formation by RNA interference and chemically induced mutations to genes expressed in the axillary meristem of Nicotiana tabacum L.

BACKGROUND: Lateral branches vigorously proliferate in tobacco after the topping of the inflorescence portions of stems for the maturation of the leaves to be harvested. Therefore, tobacco varieties with inhibited lateral shoot formation are highly desired by tobacco farmers. RESULTS: Genetic inhibition of lateral shoot formation was attempted in tobacco. Two groups of genes were examined by RNA interference. The first group comprised homologs of the genes mediating lateral shoot formation in other plants, whereas the second group included genes highly expressed in axillary bud primordial stages. Although "primary" lateral shoots that grew after the plants were topped off when flower buds emerged were unaffected, the growth of "secondary" lateral shoots, which were detected on the abaxial side of the primary lateral shoot base, was significantly suppressed in the knock-down lines of NtLs, NtBl1, NtREV, VE7, and VE12. Chemically induced mutations to NtLs, NtBl1, and NtREV similarly inhibited the development of secondary and "tertiary" lateral shoots, but not primary lateral shoots. The mutations to NtLs and NtBl1 were incorporated into an elite variety by backcrossing. The agronomic characteristics of the backcross lines were examined in field trials conducted in commercial tobacco production regions. The lines were generally suitable for tobacco leaf production and may be useful as new tobacco varieties. CONCLUSION: The suppressed expression of NtLs, NtBl1, NtREV, VE7, or VE12 inhibited the development of only the secondary and tertiary lateral shoots in tobacco. The mutant lines may benefit tobacco farmers by minimizing the work required to remove secondary and tertiary lateral shoots that emerge when farmers are harvesting leaves, which is a labor-intensive process.

DNA marker development by the allele-specific detection of powdery mildew resistance loci derived from Japanese domestic tobacco cultivar 'Kokubu'.

Japanese domestic tobacco (Nicotiana tabacum L.) cultivar 'Kokubu' shows high powdery mildew resistance controlled by recessive alleles at two loci, and these alleles have been widely used as a resource for powdery mildew resistance in tobacco breeding. However, the introduction of this trait by conventional breeding takes much work because of the requirement for test crosses with the parental strains and inoculation tests using active fungi to confirm the introduction of two recessive alleles during back-crossing. Recently, we found that powdery mildew resistance in 'Kokubu' is caused by splice site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. Here, we report DNA markers that detect mutations of the NtMLO1/2 genes based on the cleaved amplified polymorphic sequence (CAPS) or allele-specific polymerase chain reaction (AS-PCR) methods. These markers can be used as co-dominant markers that detect heterozygotes of the NtMLO genes at the seedling stage in back-crossed progenies, and will contribute to the simplification of breeding.

Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids.

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.