A Rice Semi-Dwarf Gene,Tan-Ginbozu (D35), Encodes the Gibberellin Biosynthesis Enzyme,ent-Kaurene Oxidase

A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like (KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1 to 5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2 corresponds to D35. Homozygote with null alleles of D35 showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels.     

Identification of Tobacco HIN1 and Two Closely Related Genes as Spermine-Responsive Genes and their Differential Expression During the Tobacco Mosaic Virus-Induced Hypersensitive Response and During Leaf- and Flower-Senescence

Previously we showed that the polyamine spermine (Spm) specifically leads to mitochondrial dysfunction in tobacco that is followed by the activation of salicylic acid-induced protein kinase and wound-induced protein kinase. To identify the possible downstream components of the Spm signalling pathway, we isolated Spm-responsive genes by a differential hybridization approach. This showed that the harpin-induced 1 (HIN1) gene is responsive to Spm. Genomic Southern analysis showed that HIN1 constitutes a multi-gene family and this led to the isolation of two novel HIN1 -like tobacco cDNAs that we designated as HIN9 and HIN18. Both genes are also responsive to Spm, albeit HIN18 is induced weakly compared to HIN1 and HIN9. As HIN1 is up-regulated both during the hypersensitive response (HR) generated by an incompatible plant-pathogen interaction and during senescence, we compared the expression of the three HIN1 family genes in these situations. All three were responsive to HR due to Tobacco mosaic virus infection, although HIN18 was less efficiently induced, and HIN1 and HIN18 were both strongly up-regulated during leaf- and flower-senescence. This suggests that the signalling pathways in the HR and senescence overlap somehow but are distinct. That HIN1 and its closely related genes are Spm-responsive genes also supports the idea that Spm plays a role as a signal transmitter in the HR process.     

Characterization and cloning of plasmids from the iron-oxidizing bacteriumThiobacillus ferrooxidans

More than 100 independent strains ofThiobacillus ferrooxidans were isolated from six different domestic mining sites. Although there was some variation according to sampling site, about 73% of all strains carried more than one plasmid ranging in size from about 2.0 to 30 kilobase-pairs(kb). Among these, four plasmids of 2.4, 4.7, 5.1, and 8.9 kb, designated pTNA33, pTSY91, pTSB121, and pTSB122, respectively, were cloned intoEscherichia coli plasmids. pTSB121 and pTSB122, originated from the sameT. ferrooxidans strain, showed weak homology by Southern blotting, whereas pTSB121 showed high homology with pTSY91 from a different strain. It seems that the occurrence of the plasmid homologous to pTSB121 or pTSB122 is more ubiquitous inThiobacillus. On the other hand, pTNA33 is a unique plasmid because it showed no significant homology with other plasmids.     

Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.