Hidenari Shioiri

In Planta Transformation of Kenaf Plants (Hibiscus cannabinus var. aokawa No. 3) by Agrobacterium tumefaciens

Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.

Detection and characterization of a 36-kDa peptide in C-terminal region of a 24-kDa vacuolar protein (VP24) precursor in anthocyanin-producing sweet potato cells in suspension culture.

A 24-kDa vacuolar protein (VP24) was found to accumulate in anthocyanin-producing sweet potato cells (Ipomoea batatas Lam.) in suspension culture [Nozue et al., Plant Physiol. 115 (1997) 1065]. VP24 cDNA (accession No. AB025531) encodes a 96.3-kDa large precursor protein with a C-terminal propeptide which contains the eight putative transmembrane domains. The mature VP24 is probably involved in the formation of intravacuolar pigmented globules (cyanoplasts) in highly anthocyanin-containing vacuoles, but the biological function of the C-terminal region including the putative transmembrane domains is unknown. To confirm the expression and characterize the C-terminal region in the VP24 protein precursor in the anthocyanin-producing cells, polyclonal antibodies were developed against the fusion protein, including the C-terminal region, expressed in Escherichia coli. Western blot analysis showed that a 36-kDa peptide (VP36) localized in anthocyanin-containing vacuoles was expressed under continuous illumination, but not in darkness. The expression pattern of VP36 showed high similarity to VP24. These results suggested that VP36 may be derived from the large VP24 protein precursor; it includes several of the hydrophobic domains in the C-terminal region.

M-31 mutant (virA::Tn5) of Agrobacterium tumefaciens is capable of transferring its T-DNA into the nucleus of host cell, but incapable of integrating it into the chromosome.

An avirulent mutant (M-31 strain) was produced by the transposon (Tn5) mutagenesis of Agrobacterium tumefaciens (A-208 strain). A binary vector, pIG121-Hm, containing a kanamycin resistance gene (nptII) and beta-glucuronidase (GUS) gene with an intron, was introduced into M-31 and A-208 strains. The resultant Agrobacteria were inoculated onto leaves of Kalanchoe daigremontiana and to tobacco BY-2 cells to assay GUS activity to monitor the T-DNA transfer into the nuclei of host cells. The results indicated that T-DNA was transferred into the nuclei of cells of both host plants inoculated with the M-31 mutant. The M-31 mutant strain had an insertion of Tn5 in the virA gene on its Ti plasmid. The introduction of the virA gene in the M-31 mutant complemented its avirulent phenotype. No kanamycin-resistant cells were observed when the M-31 mutant harboring the pIG121-Hm was inoculated to tobacco BY-2 cells. The M-31 mutant (virA::Tn5) seems to transfer T-DNA into the nucleus of the host cell, but is unable to integrate it to the chromosome.

Activation by Proteolysis in vivo of 60-ku Latent Polyphenol Oxidases in Sweet Potato Cells in Suspension Culture

Summary Polyphenol oxidases (PPOs) with two different molecular masses (40 ku and 60 ku) were detected in sweet potato cultured cells. The 40-ku form actually consisted of isoforms of 39 ku and 40 ku with strong activity, while the 60-ku form represented isoforms of 60 and 65 ku with little activity. The partially purified 60-ku PPO was activated by sodium dodecyl sulfate (SDS). Large amounts of 60-ku PPOs and small amounts of 40-ku PPOs were detected in subcultured cells that had been maintained in medium that contained 2,4-dichlorophenoxyacetic acid (2,4-D) and 3 % sucrose in darkness. When the subcultured cells were transferred to 2,4-D free-medium and cultured with continuous illumination, levels of 40-ku PPOs increased, with an increase in PPO activity, in the absence of SIDS. The conversion of 60-ku PPOs to the 40ku form was completely inhibited by inhibitors of serine protease. These results suggest that the active 40-ku PPO is generated by proteolytic cleavage of the 60-ku form and that such cleavage by an endogenous serine protease might be initiated when the cells are cultured in 2,4-D-free medium with continuous illumination. Such proteolysis in vivo might be a mechanism for the activation of latent PPOs in these plant cells.

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome.