Yusuke Kosugi

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.

2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers

Abstract2-Aminooxyisobutyric acid (AOIB) has a partial structure of aminooxyacetic acid (AOA) in its whole structure, and resembles 2-aminoisobutyric acid (AIB) in their tetrahedral structures. Both AOA and AIB are inhibitors of ethylene biosynthesis; AOA inhibits the action of 1-aminocyclopropane-1-carboxylate (ACC) synthase and AIB inhibits that of ACC oxidase. The present study showed that AOIB inhibited the in vitro activities of both ACC synthase and ACC oxidase, which were synthesized heterologously in E. coli cells from corresponding carnation cDNAs, and the magnitudes of inhibition were similar to those caused by AOA and AIB; AOIB and AOA at 0.1 mM inhibited ACC synthase action by 75%, and AOIB and AIB at 10 mM inhibited ACC oxidase action by 16.3 and 22.5%, respectively. AOIB at 1 mM caused 91.5% reduction of maximum ethylene production rate as compared to the control in cut ‘Excerea’ carnation flowers undergoing senescence, thereby lengthening their vase life to 7 d from 3 d of the control flowers. The inhibition by AOIB was probably caused by its action resembling AOA, but not AIB. AOIB also extended significantly the vase life of cut flowers of ‘Pax’ carnation, and tended to do so in ‘Primero Mango’ carnation. The present findings suggest the potential of AOIB as a new preservative for carnations and other ornamentals in which ethylene plays a key role in the induction of senescence.

Escherichia coli -Based Expression and In Vitro Activity Assay of 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase and ACC Oxidase

1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase are key enzymes in the ethylene biosynthetic pathway in plant tissues, and in vitro assay of their activities is indispensable for analysis, especially, for studying the action mechanism of inhibitors of ethylene biosynthesis. The enzymes can be obtained from plant tissues that are producing ethylene abundantly, such as ripening fruit- and senescing flower tissues, but it is necessary to separate the enzymes from co-extracted ACC by partial purification, making the procedure laborious and time-consuming. Here, we describe the production of the enzymes in Escherichia coli cells from corresponding cDNAs, and the procedures for assay of activities of the enzymes.

Textural and Compositional Changes of Stored Iceberg Lettuce in Relation to Harvest Season and Storage Condition

The quality and storability of iceberg lettuce (Lactuca sativa L.) is dependent on growing and storage conditions, which are not completely known. Lettuce was planted at approximately monthly intervals from Sept. 2006 to Feb. 2007. Crops were harvested at approximately monthly intervals from Nov. 2006 to May 2007 and held in storage at 3 and 13°C. Effects of harvest month and storage condition on storage quality—i.e., changes in textural quality, sugar and organic acid contents—as well as shelf-life in iceberg lettuce were examined. Higher values for puncture force and breaking energy and increased sugar content were found in the cooler than in the warmer months. Lower leaf crispness was recorded after one week of storage with a greater degree at 13°C. Total soluble sugar content declined by about 56% and 32% after one week of storage at 13°C in the outer and inner leaves, respectively. Organic acid content did not show any consistent trend. At the end of the harvest season, shelf-life declined by 40% and 30% during storage at 3 and 13°C, respectively. Results indicate that low temperature during growth and storage could ensure a longer shelf-life along with a higher quality of iceberg lettuce.

Cloning and expression analysis of a cDNA partially encoding glutamate dehydrogenase in lettuce during storage

The changes in ammonia content as well as activity and gene expression of glutamate dehydrogenase (GDH; EC 1.4.1.2) were investigated in lettuce during storage. GDH amination activity increased with the increases in ammonia content in the outer leaf portion after 24 h of storage. GDH amination activity was substantially higher than deamination activity. The isolated partial cDNA clone referred to as LsGDH (Lactuca sativa glutamate dehydrogenase; AB334207) consisted of 757 nucleotides and was highly homologous with the GDH genes of other plants. Although the transcript of LsGDH was found in both the outer and inner leaves, the level of transcript gradually increased in the outer leaves with the progress of storage, but was only expressed in the inner leaves when higher enzyme activity was observed. Results suggest that GDH expression in lettuce is controlled by tissue specific manner and/or multiple levels of regulations.