Nobumaro Kawashima

The alkaloid contents of sixty Nicotiana species

Abstract The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F2 progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F1 hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed.

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.

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.

Two labdane diterpenoids from Nicotiana setchellii

Abstract Two new labdanoids isolated from Nicotiana setchellii have been identified as labda-7,13 E -dien-15-ol and labda-8,13 E -dien-15-ol.

Origin of Nicotiana tabacum detected by primary structure of fraction I protein.

Nicotiana tabacum is believed to have arisen after hybridization of Nicotiana sylvestris with a species in the Tomentosae section of the genus Nicotiana. Recent biochemical experiments have confirmed the conclusions from previous cytogenetic studies that N. sylvestris was the maternal parent and have indicated that Nicotiana tomentosiformis was the paternal parent. However, these studies did not take into account the possibility that a new species of Nicotiana, called K-12, discovered in South America in 1968, could also have been one of the parents. Fraction I proteins were purified from N. tabacum and its putative progenitors, and separated into large and small subunits. Chymotryptic peptides of each subunit were analyzed by ion exchange column chromatography with a gradient elution system. Among 38 resolved peaks of the large subunits, 2 peaks were found to be different among the putative species. Since only N. sylvestris showed an identical chromatogram with N. tabacum, N. sylvestris was concluded to be the maternal progenitor, as the genetic information for the large subunit of Fraction I protein was known to be inherited by the cytoplasmic mode. On the other hand, the small subunit of Fraction I protein is inherited by the Mendelian mode and therefore N. tabacum, an allopolyploid, could be expected to contain two types of small subunits, one derived from N. sylvestris and the other from a paternal progenitor. N. sylvestris lacks two of the 25 chymotryptic peptides of the small subunit of N. tabacum. Among 3 putative paternal progenitors, these two peaks appeared only in N. tomentosiformis, but not in Nicotiana otophora or K-12. Thus, N. tomentosiformis was concluded to be a paternal progenitor of N. tabacum. The conclusion was verified by comparing chymotryptic peptides of small subunits from three amphidiploids of N. sylvestris crossed with N. tomentosiformis, N. sylvestris crossed with N. otophora snd N. sylvestris crossed with K-12. The analytical results showed that only the progeny of N. sylvestris crossed with N. tomentosiformis contained the same small subunits as N. tabacium.

Two labdane diterpenoids from Nicotiana raimondii

Abstract Two new labdanoids from Nicotiana raimondii have been identified as (12 E ) - 7β - hydroxylabda - 8(20),12,14 - triene and 7β - hydroxy - labda - 8(20),13(16),14 - triene.

Similarities and differences in the primary structure of fraction I proteins in the genus Nicotiana.

Fraction I proteins were purified from 30 species of the genus Nicotiana, and separated into large and small subunits which in all cases displayed the same relative ratio. Tryptic peptides of each subunit which were soluble at pH 2.2 were analyzed by ion-exchange column chromatography with a gradient elution system. N. tabacum was used as a standard for comparison, 27 peptides being resolved from the large subunit and 23 for the small subunit. The composition of Fraction I proteins from other Nicotianas ranged from no differences (7 species) to as many 13 differences distributed in both the large and small subunits. The distribution of differences allowed a chemical taxonomic classification of the genus to be made which was closely similar (with only two excpetions) to the classical taxonomy. The results confirm a previous concept that mutation of the small subunit coded by nuclear DNA is much more frequent than mutation of the large subunit coded by chloroplast DNA. The differences also serve as unique phenotypic markers for both nuclear and chloroplast DNA genes coding for Fraction I protein. The markers will be of great utility in further studies of evolution, transfer of genetic information by parasexual hybridization and mode of chloroplast DNA mutation.

Production of Nicotiana tabacum x Nicotiana acuminata hybrid by ovule culture

Using a conventional sexual crossing technique, Nicotiana tabacum x N. acuminata was not produced. After the fertilized ovules were cultured for 20 days in a liquid Nitsch H medium, germination was observed. The roots grew rapidly but leaves did not. However, plantlets were produced in an H medium containing Benzyladenine or Kinetin (0.01–0.1 mg/l). The plantlets grew and flowered in a greenhouse. The chromosome number of the hybrid was 36 and its morphological characteristics were intermediate between those of parental species.

Changes in glycerolipids and their fatty acid composition during maturation of tobacco seeds

Abstract Triacylglycerol, which was one of the minor lipid components in immature seeds of tobacco, accumulated dramatically between 7 and 27 days after flowering and, in mature seeds at 37 days, the fatty acid methyl esters of the triacylglycerols comprised 96.3% of those of the total lipids. Diacylglycerols and sterol ester also increased significantly during seed development. Phosphatidylcholine and phosphatidylethanolamine, which were major components in immature seeds, decreased constantly with increasing maturation as well as the quantities of phosphatidylinositol and phosphatidylglycerol. Monogalactosyldiacylglycerols, digalactosyldiacylglycerols and sulfoquinovosyldiacylglycerols also decreased and disappeared in mature seeds. In the triacylglycerols the percentages of palmitate, stearate and linolenate fell with increasing seed age, while that of linoleate increased up to 75.3% in mature seeds. A similar trend was observed in the fatty acid composition in the diacylglycerols and sterol ester. Generally, in the phospholipids the proportions of linoleate and linolenate decreased with concomitant increases of stearate and oleate.