Akira Koiwai

Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme in the regulation of intracellular methylation reactions. To investigate the role of SAHH in methylation reactions and morphogenesis in planta, we have made transgenic plants expressing antisense RNA of tobacco SAHH. The transgenic plants displayed distinct morphological changes including a floral homeotic change. We hypothesized that the changes were caused by increased levels of cytokinin. In those transgenic plants, we observed that a repetitive DNA sequence appeared less methylated than controls. We speculated that altered gene expressions by the hypomethylation of DNA might be involved in the changes.

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)+ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type α genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH2 and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type α genomic clone is a functional protein exhibiting RNA N-glycosidase activity.

Molecular cloning of a cDNA clone for tobacco lipid transfer protein and expression of the functional protein in Escherichia coli

A cDNA clone encoding a lipid transfer protein (LTP) was isolated from tobacco by screening a library with a PCR‐amplified spinach LTP gene, DNA sequence analysis showed a large open reading frame (344 bp) encoding a polypeptide of 114 amino acids. The first 23 amino acids of the deduced protein have the characteristics of a signal peptide for protein secretion or targeting into dense microbody‐like vesicles. The cDNA clone was then inserted into an expression vector, pMAL, and expressed in E. coli as a fusion with the maltose binding protein (MBP). The MBP‐LTP fusion protein was purified to homogeneity and subjected to factor Xa cleavage to yield the LTP domain. A lipid transfer assay demonstrated that the resulting LTP was functional. The availability of the expression system in E. coli will facilitate the elucidation of in vivo function(s) of plant LTPs.

The fatty acid composition of seeds and leaves of Nicotiana species

Abstract Fatty acid analyses of seeds in 62 Nicotiana species and leaves in 56 Nicotiana species are presented. The total fatty acid content on a dry wt basis ranged from 25 to 40 %of seeds and from 2.1 to4.4% of green leaves. Linolenate was the dominant fatty acid in the leaves of all species studied, comprising 50–63% of the total fatty acid content. In seeds of most species linoleate predominated, constituting 69–79% of the total fatty acid content. Fourteen of 21 species in the section Suaveolentes and one species in the section Noctiflorae had relatively high proportions (10–38%) of linolenate. In two linolenate-rich species studied, linolenate was the major fatty acid of triacylglycerols which predominated in the seed lipids.

Inducible expression by plant hormones of S-adenosyl-l-homocysteine hydrolase gene from Nicotiana tabacum during early flower bud formation in vitro

Abstract cDNA clones corresponding to genes induced during the early stage of flower bud formation in thin cell layer (TCL) cultures of tobacco were isolated by differential screening. Sequence analysis showed that one of the clones encoded S -adenosyl- l -homocysteine hydrolase (SAHH), which is known to be an enzyme regulating intracellular transmethylation reactions. Recently, it was also found to be a cytokinin-binding protein. An SAHH gene was isolated by screening a tobacco genomic library with the cDNA as a probe. Northern blot analysis showed that expression of the SAHH gene was greatest in pistils and roots, and could be induced by kinetin and IAA. Kinetin and IAA are required for in vitro flower differentiation in TCL culture. The promoter sequence of the gene was fused to the β-glucuronidase (GUS) reporter gene and introduced in suspension-cultured cells, which rendered expression of GUS inducible by kinetin. Possible involvement of SAHH in regulation of gene expression, cytokinin signal transduction and flower differentiation is discussed.

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization

Two expression vectors were constructed to produce a putative mature α‐pokeweed antiviral protein (α‐PAP) in Escherichia coli with its NH2‐ and COOH‐terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2‐terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N‐glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.

Changes in the amino acid composition of tobacco cells in suspension culture

Abstract Changes in the content of free and protein amino acids of tobacco cells in suspension culture during the growth were determined. The principal free amino acids throughout the culture period were glutamine, asparagine, and γ-aminobutyric acid. Glutamic acid, glycine, and lysine decreased in the lag period, followed by the increase in the log period, and then decreased towards the stationary phase. By contrast alanine, serine, valine, leucine, and proline increased in the lag period, followed by the decrease in the log period. The contents of glutamic acid and hydroxyproline in the protein fraction changed markedly during the growth. The glutamic acid content increased in the early period of the log phase and then gradually decreased whereas the hydroxyproline content decreased in the log period and then abruptly increased.

Sucrose esters from the surface lipids of Nicotiana cavicola

Three sucrose esters were isolated and identified for the first time from the surface lipids of Nicotiana cavicola. They contained acetic acid and such branched short-chain fatty acids as 5-methylhexanoic, 5-methylheptanoic, and 6-methylheptanoic acids. The structures of the sucrose esters were 6-O-acetyl-4-O-acyl-alpha-D-glucopyranosyl-4,6-O-diacetyl-3-O-acyl-beta- D- fructofuranoside, 4-O-acyl-alpha-D-glucopyranosyl-1,4-O-diacetyl-3-O-acyl-beta-D-fructofur anoside and 6-O-acetyl-4-O-acyl-alpha-D-glucopyranosyl-4-O-acetyl-3-O- acyl-beta-D-fructofuranoside.

New Types of Glycolipids from the Surface Lipids of Nicotiana umbratica

The surface lipids of Nicotiana umbratica contained several varieties of glycolipids. Three types of glucose esters and five types of sucrose esters were isolated and identified from this species. These glycolipids contained short-chain fatty acids such as acetic, methylpropionic, methylbutyric, 3-methylpentanoic, 4-methylpentanoic and methylhexanoic acids. The acetylated positions of each compound were determined by 2-D NMR, using the HMBC technique. The structures of the glucose esters were 1,6-di-O-acetyl-2,3-4-tri-O-acyl-alpha-D-glucopyranose (I), 6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranose (II) and 2,3,4-tri-O-acyl-alpha, beta-D-glucopyranose (III). The structures of the sucrose esters were 6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-1,3-di-O-acetyl-beta- D- fructofuranoside (IV), 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-1,3-di-O-acetyl-beta-D-fructofur anoside (V), 6-O-acetyl-2,4-di-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D- fructofuranoside (VI), 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D-fructofuranosi de 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D-fructofuranosi de (VII) and 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-beta-D-fructofuranoside (VIII). Among them, I, IV, V and VI were isolated for the first time from plants of the family Solanaceae. These newly isolated glycolipids were inhibitory against barnyardgrass growth.