Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells

Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.Abbreviations IAA indole-3-acetic acid - LMT S-adenosylmethionine: licodione 2-O-methyltransferase - CHS chalcone synthase     

Flavonoids of Leguminous Plants: Structure, Biological Activity, and Biosynthesis

Received 25 August 2000/ Accepted in revised form 2 October 2000     

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F₂ population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

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Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract.

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned.     

Complex feeding tracks of the sessile herbivorous insect Ophiomyia maura as a function of the defense against insect parasitoids

Because insect herbivores generally suffer from high mortality due to their natural enemies, reducing the risk of being located by natural enemies is of critical importance for them, forcing them to develop a variety of defensive measures. Larvae of leaf-mining insects lead a sedentary life inside a leaf and make conspicuous feeding tracks called mines, exposing themselves to the potential risk of parasitism. We investigated the defense strategy of the linear leafminer Ophiomyia maura Meigen (Diptera: Agromyzidae), by focusing on its mining patterns. We examined whether the leafminer could reduce the risk of being parasitized (1) by making cross structures in the inner area of a leaf to deter parasitoids from tracking the mines due to complex pathways, and (2) by mining along the edge of a leaf to hinder visually searching parasitoids from finding mined leaves due to effective background matching of the mined leaves among intact leaves. We quantified fractal dimension as mine complexity and area of mine in the inner area of the leaf as interior mine density for each sample mine, and analyzed whether these mine traits affected the susceptibility of O. maura to parasitism. Our results have shown that an increase in mine complexity with the development of occupying larvae decreases the probability of being parasitized, while interior mine density has no influence on parasitism. These results suggest that the larval development increases the host defense ability through increasing mine complexity. Thus the feeding pattern of these sessile insects has a defensive function by reducing the risk of parasitism.     

Triterpenoid biosynthesis in tissue cultures of Glycyrrhiza glabra var. glandulifera

The incorporation of [1-¹⁴C]acetate and [2¹⁴ C]mevalonate into free and esterified triterpen-3-ols was examined in original plant organs and tissue cultures of Glycyrrhiza glabra var. glandulifera. Both substrates labeled -amyrin, an oleanane-type triterpene, and cycloartenol and 24-methylenecycloartanol, both of which are intermediates of phytosterol biosynthesis. The label in esterified triterpenes was distributed mainly in phytosterol intermediates, but not in -amyrin. The ratio of amyrin formation among the three triterpenes from [2-¹⁴C]mevalonate was relatively high in stolon segments and in root cultures, but negligible in callus cultures. Administration of a specific inhibitor of squalene-2, 3-epoxide:cycloartenol (lanosterol) cyclase caused a marked increase of -amyrin synthesis in root suspension cultures, and of 24-methylenecycloartanol synthesis in cell suspension cultures, from [2-¹⁴C]mevalonate.Abbreviations GL glycyrrhizin - GLA glycyrrhetic acid - MVA mevalonic acid - CCI squalene-2, 3-epoxide:cycloartenol cyclase inhibitor This paper is Part 72 in the series Studies on Plant Tissue Cultures from the laboratory at Kitasato University. For Part 71, see Furuya T, Koge K, Orihara Y (submitted for publication).     

Phosphorylation of D-glucosamine by rat liver glucokinase.

D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.     

Diploid male production in a rare and locally distributed bumblebee, Bombus florilegus (Hymenoptera, Apidae)

The European bumblebee B. terrestris was recently introduced in Japan for agricultural purposes and has now become naturalized. The naturalization of this exotic species may have great detrimental effects on closely related native Japanese bumblebees. The Japanese bumblebee Bombus florilegus is a rare and locally distributed species found in the Nemuro Peninsula of Hokkaido, Japan. In order to assess its population genetics, we estimated the genetic structure of B. florilegus in 16 breeding colonies (queen, workers, and males) and 20 foraging queens by analyzing microsatellite DNA markers. Of the 36 queens analyzed by genotyping and dissection, 32 had been inseminated by a male. The remaining 4 had not been inseminated at all. Of the 4 nonmated queens, one was triploid. Diploid males were found in 4 breeding colonies. Based on the microsatellite data, it appears that B. florilegus has low reproductive success. Since matched mating and nonmating within local populations are high, the extinction risk is correspondingly higher. Our results suggest that conservation of the Japanese B. florilegus is required in order to protect it from both habitat destruction and the naturalization of alien species. Received 19 July 2007; revised 13 October 2007; accepted 15 October 2007.