Toshihito Yoshioka

Expression of genes responsible for ethylene production and wilting are differently regulated in carnation (Dianthus caryophyllus L.) petals.

Carnation petals exhibit autocatalytic ethylene production and wilting during senescence. The autocatalytic ethylene production is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes, whereas the wilting of petals is related to the expression of the cysteine proteinase (CPase) gene. So far, it has been believed that the ethylene production and wilting are regulated in concert in senescing carnation petals, since the two events occurred closely in parallel with time. In the present study, we investigated the expression of these genes in petals of a transgenic carnation harboring a sense ACC oxidase transgene and in petals of carnation flowers treated with 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS). In petals of the transgenic carnation flowers, treatment with exogenous ethylene caused accumulation of the transcript for CPase and in-rolling (wilting), whereas it caused no or little accumulation of the transcripts for ACC oxidase and ACC synthase and negligible ethylene production. In petals of the flowers treated with DPSS, the transcripts for ACC synthase and ACC oxidase were accumulated, but no significant change in the level of the transcript for CPase was observed. These results suggest that the expression of ACC synthase and ACC oxidase genes, which leads to ethylene production, is differentially regulated from the expression of CPase, which leads to wilting, in carnation petals.

Role of abscisic acid in flood-induced secondary aerenchyma formation in soybean (Glycine max) hypocotyls.

Abstract Phellogen (cork cambium) usually produces cork tissue, but when flooded it produces secondary aerenchyma, comprising living cells with non-suberized walls in the stems, roots, and root nodules of some Fabaceae. In the cell walls of cork tissues, the plant hormone abscisic acid (ABA), promotes suberin deposition. Thus, ABA may decrease in flooded tissues, where secondary aerenchyma cells are developing. Here, we investigated whether ABA is involved in the formation of aerenchyma in soybean (Glycine max) hypocotyls when flooded. Hypocotyls flooded with water produced a large amount of secondary aerenchyma, and were highly porous. On the other hand, application of 1.0 μM ABA suppressed the enlargement of phellogen-derived cells, thereby suppressing subsequent gas space formation, and then almost completely inhibited aerenchyma development. Berberine-aniline blue staining indicated that not only elongated cells in the secondary aerenchyma but also packed cells, which were formed under flooding with ABA, contained no suberized cell walls. Compared to non-flooded plants, the endogenous ABA concentration in the flooded hypocotyls was decreased to 50% within 24 hr, and the low level was maintained for at least 72 hr. In addition, phellogen developed at 48 hr after flooding and secondary aerenchyma was observed at 72 hr. These results indicate that secondary aerenchyma formation requires a decrease in negative regulator ABA in soybean plants, that is, ABA inhibits elongation of cells derived from phellogen in secondary aerenchyma formation such as internodal cell elongation of floating rice stems.

Cloning of a Novel Dehalogenase from Environmental DNA

Cloning of pceA, the gene of tetrachloroethene (PCE)-reductive dehalogenase, was undertaken from environmental DNA. Two genes were amplified using PCR primer deduced from pceA. Functional expression of these genes was unsuccessful in Escherichia coli, but PceA1 synthesized in vitro was enzymatically active. In recombinant E. coli, PceA1 formed a complex with host DnaK and caused filamentous growth.

Anoxia-enhanced expression of genes isolated by suppression subtractive hybridization from pondweed (Potamogeton distinctus A. Benn.) turions

Pondweed (Potamogeton distinctus A. Benn.), a monocot aquatic plant species, has turions, which are overwintering buds forming underground as an asexual reproductive organ. Turions not only survive for more than one month but also elongate under strict anoxia, maintaining high-energy charge by activation of fermentation. We cloned 82 cDNA fragments of genes, that are up-regulated during anoxic growth of pondweed turions, by suppression subtractive hybridization. The transcript levels of 44 genes were confirmed to be higher under anoxia than those in air by both Northern blot analysis and a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method. A homology search for their nucleotide sequences revealed that some of them are highly homologous to known sequences of genes from other plants. They included alcohol dehydrogenase, pyruvate decarboxylase (PDC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vacuolar H+-translocating pyrophosphatase and a plasma membrane intrinsic protein. Time courses of transcript accumulation of some genes under anoxia were different from those in air. The activity of PDC increased under anoxic conditions but the activities of GAPDH and pyrophosphatase remained constant after anoxic treatment. Anoxically up-regulated genes are possibly involved in physiological events to control energy production, pH regulation and cell growth under anoxia. These results suggest that transcriptional regulation of these genes serves as an essential part of survival and growth of pondweed turions under anoxia.

Stimulated ethylene production in tobacco (Nicotiana tabacum L. cv. Ky 57) leaves infected systemically with cucumber mosaic virus yellow strain

Ethylene production was stimulated in the leaves of tobacco (Nicotiana tabacum L. cv. Ky 57) infected systemically with cucumber mosaic virus yellow strain (CMV-Y). A transient peak of ethylene production per fresh-weight base appeared 2 weeks after inoculation when the mosaic symptoms of green, yellow and white sectors covered about 40% or the leaf area. The increase in ethylene production was accompanied by the increase in 1-aminocyclopropane-1-carboxylate (ACC) content and activities of ACC synthase and ACC oxidase in systemically-infected leaves. Application of aminooxyacetic acid or 1,10-phenanthroline suppressed the multiplication of the virus and development of mosaic symptoms besides ethylene production, suggesting a causal relationship of the stimulated ethylene production in the symptoms development. A partial cDNA encoding a putative ethylene receptor was isolated by RT-PCR from virus-infected tobacco leaves. The abundance of mRNA corresponding to the cDNA increased during mosaic symptoms development in tobacco leaves after systemic infection with the virus.

1,1-Dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) does not inhibit the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase obtained from senescing carnation (Dianthus caryophyllus L.) petals

The effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase isolated from senescing carnation petals were investigated. In contrast to a previous proposal, DPSS at 1 mM did not inhibit the in vitro activity of ACC oxidase. It was confirmed that DPSS does not inhibit ACC synthase activity. DPSS probably does not exert its inhibitory action on ethylene production by a direct action on ACC oxidase and ACC synthase, but by some unknown action.

Induction by ozone of ethylene production and an ACC oxidase cDNA in rice (Oryza sativa L.) leaves

Exposure to ozone at 1 µl l−1 for 6 h induced ethylene production in rice (Oryza sativa L. cv. Hitomebore) leaves. The stimulation of ethylene production was detectable 2 h after the start of the exposure to ozone, and lasted for 6 h after the exposure. A 429-bp cDNA fragment encoding ACC oxidase was obtained by RT-PCR from ozone-treated rice leaves. Its nucleotide sequence and deduced amino-acid sequence had 97.2% and 94.4% identity, respectively, to those of OS1A1COX, which was previously obtained from deepwater rice. The abundance of the cDNA increased in accordance with the induction of ethylene production by the exposure to ozone.

Action of 1,1-Dimethyl-4-(Phenylsulfonyl) Semicarbazide (DPSS), A New Antisenescence Preservative for Cut Carnation Flowers

Increasing flower’s longevity by blockage of the onset of senescence has economical importance. Currently, STS (silver thiosulfate complex anion), is being used as an anti-senescence preservative in the cut-flower industry. But, STS contains a heavy metal ion (Ag+) and has a potential environmental hazard. Therefore, it is desired to have alternatives to STS, which are safe for organisms and have a less risk for environmental pollution. DPSS is a novel preservative for cut carnation flowers, which were developed recently [1] and has just come to the market in Japan. Its activity to retard carnation flower senescence is compatible to or more than that of STS. DPSS is safe for organisms including mammals, because its oral and dermal administration causes no acute toxicity in mice and exerts no mutagenic effects by Ames test.