Sun Hi Lee

Effects of spermine on ethylene biosynthesis in cut carnation (Dianthus caryophyllus L.) flowers during senescence

Summary To investigate the relationship between polyamine and ethylene during senescence of cut carnation ( Dianthus caryophyllus L.) flowers, we studied the effects of spermine on ethylene biosynthesis. Spermine delayed the senescence of cut carnation flowers and reduced ethylene production, endogenous 1-aminocy-clopropane-l-carboxylic acid (ACQ content, and the activities and transcript amounts of ACC synthase and ACC oxidase in petals. Methylglyoxal bis-(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, elevated ethylene production, increased activities and amounts of transcripts for ACC synthase and ACC oxidase, and shifted the climacteric pattern of ethylene production ahead by 1 day. However, endogenous ACC content was not increased in the petals of MGBG-treated flowers because of the high activity of ACC oxidase. Spermine also inhibited MGBG-induced ethylene production by decreasing the activities and amounts of transcripts for ACC synthase and ACC oxidase. The accumulation of transcripts for ACC synthase and ACC oxidase in MGBG-treated and in climacteric control petals was correlated with the increase of these enzyme activities. By comparing ethylene production with the changes of endogenous polyamine levels from control and MGBG- or spermine-treated petals during the entire incubation period, it was suggested that endogenous polyamines possibly suppress ethylene production.

Characterization and expression of two members of the S-adenosylmethionine decarboxylase gene family in carnation flower

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50) is one of the key enzymes in polyamine biosynthesis, and the product of its catalytic reaction, decarboxylated S-adenosylmethionine (dcSAM), serves as an aminopropyl donor in the biosynthesis of spermidine and spermine. In order to provide information on the structure and regulation of SAMDC, we have isolated and sequenced two different SAMDC cDNA clones from carnation petals. The nucleotide sequences of CSDC9 and CSDC16 show 78.3% identity, and the deduced amino acid sequences show 81.7% identity and 86.5% similarity [12]. There are several regions with highly conserved sequences among SAMDC cDNAs of potato, spinach, periwinkle, man and yeast. These conserved regions include a cleavage site for the processing of SAMDC proenzyme and a putative PEST sequence that may be relevant to the rapid degradation of SAMDC protein. Carnation SAMDC cDNAs have long transcript leaders of 472 bp and 502 bp for CSDC9 and CSDC16, respectively. Both sequences contain short upstream open reading frames (uORFs) in their 5′ -untranslated regions. The CSDC9 uORF is 54 amino acids from 152 to 317 while the corresponding sequence in CSDC16 is 52 amino acids located from 156 to 314 in each 5′-untranslated region. The nucleotide sequences of uORFs in CSDC9 and CSDC16 were 89.9% identical. In vitro transcription/translation experiments showed: (1) each proenzyme of both cDNAs of SAMDC was converted to two polypeptides consisting of a large subunit (calculated as 31544 Da and 32537 Da, respectively) and a small subunit (calculated as 9704 and 9041 Da, respectively) after 20 min of translation; (2) the processing occurs rapidly during the translation of protein. But once the translation process is stopped accumulation of the subunits slows and never reaches completion even after 300 min. The processing of carnation SAMDC enzyme is not stimulated by putrescine in in vitro transcription/translation reaction.

Isolation and characterization of a jasmonic acid carboxyl methyltransferase gene from hot pepper(capsicum annuum L.)

Methyl jasmonate, the methyl ester of jasmonic acid, is a volatile plant hormone that acts as an important cellular regulator, mediating diverse developmental processes and defense responses. Methyl jasmonate is synthesized by methylation of jasmonic acid; this reaction is catalyzed by jasmonic acid carboxyl methyltransferase (JMT). Although JMT cDNA had previously been described only forArabidopsis thaliana, here we used PCR to isolate it fromCapsicum annuum L The 389-amino-acid sequence deduced for theJMT gene showed 92% identity to that fromA. thaliana. Southern blot analysis revealed thatJMT is present in the genome as two copies. Our preliminary northern blot detected no JMT transcript, but, through RT-PCR and subsequent Southern blot analysis of products using genespecific probes, we found that transcript levels increased after leaf-wounding. Likewise, 10 µM methyl jasmonate inducedJMT gene expression in leaves. Transcription levels began to increase 10 min after wounding, and were maintained for 1 to 4 h. Moreover, expression of theCaJMT andPIN2 genes was increased by both wounding and MeJA applications, but was not enhanced by treatment with H2O2.

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco.

The expression of CSDC9 encoding S‐adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5′‐flanking β‐glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5′‐flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis‐regulatory elements required for pollen‐specific transcription were located in the upstream region between −273 and −158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen‐specific expression (GTGA and AGAAA). In addition, the first 5′‐leader intron was necessary for tissue‐specific expression.

Effect of wounding and chemical treatments on expression of the gene encoding cinnamate-4-hydroxylase incamptotheca acuminata leaves

The phenylpropanoid pathway plays an important role when plants are exposed to environmental stresses, such as wounding or pathogen attack. Its activity leads to the production of lignin, flavonoids, and phytoalexins. Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of cinnamic acid to p-coumaric acid. We isolatedC4H cDNA fromCamptotheca acuminata and investigated the expression pattern of the C.acuminata C4H (CaC4H) gene following stress treatments. A search against the BLOCKS database of conserved protein motifs indicated that CaC4H shares common features with C4Hs from other species. C4H transcripts accumulated in the leaves in response to mechanical wounding or the application of molecules involved in the stress response, i.e., ethylene, methyl jasmonate, and hydrogen peroxide. Interestingly, the application of aminoethoxyvinylglycine, salicylic acid, or diphenylene iodonium, which are biosynthetic inhibitors of ethylene, methyl jasmonate, and hydrogen peroxide, respectively, did not inhibit this wound-induced expression. Based on these results, we suggest that C4H functions in response to various stresses in Gacuminata leaves.

Characterization and light-induced expression of theS-adenosylmethionine decarboxylase gene fromIpomoea nil

We screened a genomic library of Japanese morning glory(Ipomoea nil) and isolated theMGSDCgi clone (GenBank Accession Number U64927). The genomic clone comprised four exons and three introns.MCSDCgi contained a 660-bp 5′-untranslated region (UTR), a 1089-bp coding region of 362 amino acids, and a 209-bp 3′-UTR. A TATA box sequence (TATATAA) was found at position -29. The transcript leader of thisSAMDC gene contained a short, upstream open reading frame (uORF) of 51 amino acids, which was interrupted with a 107-bp intron. Two other introns were positioned upstream of the uORF in the 5’-UTR. Six-day-old seedlings were grown under a 12-h light/dark cycle, then treated with white light The amount ofSAMDC transcript dramatically increased from the first hour after light exposure, then returned to a basal level.SAMDC mRNA was accumulated more under high rather than low irradiance. These results suggest a relationship between photo-response and the physiological role of polyamines (PAs). The level ofSAMDC mRNA was increased only by brassinosteroids, not by any other plant hormone such as kinetin, IAA, ABA, and GA. This hormonal response may have replaced the effect of light onSAMDC gene expression in the dark. PAs decreased theSAMDC mRNA level, which could then be restored by their inhibitors. This indicates thatSAMDC gene expression is regulated by its cellular contents.

Generation of active oxygen species (AOS) and induction of β-Glucanase activity by fungal elicitor xylanase in the suspension cultured cells of Tobacco

It was investigated that active oxygen species (AOS) involved in the plant defense responses induced by fungal elicitor xylanase. When xylanase from the fungusTrichoderma viridae was treated to tobacco suspension cultured cells as an elicitor, β-glucanase activity was increased markedly. Lignin biosynthesis was also increased and peaked at 72 h after the treatment with xylanase. The treatment of H2O2 also dramatically increased β-glucanase activity at 24 h, which was much earlier than that of xylanase did. Using lucigenin-and luminol-dependent chemiluminescence, the effects of xylanase on oxidative burst were examined. Superoxide anion (O2) production was peaked at 40 h and 52 h after xylanase treatment and hydrogen peroxide (H2O2) release was peaked at 44 h and 56 h, suggesting H2O2 burst was followed by O2 generation. The scavengers of AOS, n-propyl gallate (PG) and mannitol, inhibited xylanase-induced β-glucanase activity by 85% and 50%, respectively. The activity of superoxide dismutase (SOD), which catalyzes the dismutation of O2 to H2O2, began to increase from 24 h and reached to maximum at 48 h after xylanase treatment. Pretreatment of N,N,-diethyldithiocarbamate (DDC), known as a SOD inhibitor, caused the inhibition of H2O2 generation by 80% and reduced the β-glucanase activity by 60%. Treatment of 2,5-norbonadiene (NBD), a specific ethylene-action inhibitor, did not have any significant effect on xylanase-induced β-glucanase activity. This result suggested that ethylene did not involve in xylanase-induced response. Our results strongly suggest that the AOS generation is an essential component in plant defense response, in which cell wall degrading enzyme, glucanase, contributes to remove the necrotic tissue induced by pathogens.

Biochemical characteristics ofS-adenosylmethionine decarboxylase from carnation (Dianthus caryophyllus L.) petals

We have partially purified S-adenosylmethionine decarboxylase (EC 4.1.1.50, SAMDC) from carnation (Dianthus caryophyllus L.) petals and generated polyclonal antibodies against CSDC 16 protein (Leeet al., 1996) overexpressed inE. coli. The protein has been purified approximately 126.8 fold through the steps involving ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephacryl S-300 gel filtration. Its molecular mass was 42 kDa in native form and we could also detect a band of 32 kDa molecular mass on SDS-PAGE in western blot analysis using the polyclonal antibodies. The Km value of this enzyme forS-adenosylmethionine was 26.3 μM. The optimum temperature and pH forS-adenosylmethionine decarboxylase activity were 35°C and pH 8.0, respectively. Putrescine and Mg2+ had no effects on the activation of the enzyme activity. Mg2+ did not have any significant effects on the enzyme activity. SAMDC activity was inhibited by putrescine, spermidine and spermine. Methylglyoxal bis-(guanylhydrazone) (MGBG), carbonyl reagents such as hydroxylamine and phenylhydrazine, and sulfhydryl reagent such as 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB) were effective inhibitors of the enzyme. However, isonicotinic acid hydrazide known as an inhibitor of 5′-pyridoxal phosphate (PLP) dependent enzyme activity had no significant effect on the enzyme activity. These results and our previously reported results (Leeet al., 1997b) suggest thatS-adenosylmethionine decarboxylase is a heterodimer, αβ, and some carbonyl group and sulfhydryl group are involved in the catalytic activity.

Surface polishing of three dimensional micro structures

A new polishing technique for three dimensional micro structures using magnetorheological (MR) fluid is presented. Among various fabrication technologies of micro devices, some processes such as electroplating and wet etching which are widely used, give usually rough surface. However, there has been no polishing technique useful to micro structures, while various existing polishing methods are applicable only to flat surfaces or macro scaled structures. In this study, three dimensional micro channel structures generated by copper electroplating and silicon wet etching are polished efficiently using MR fluid. As a result of polishing of both structures, the average surface roughness cuts down to more than an order with little change of original geometries and the performance of both structures is noticeably improved.

A 273-bp promoter region is responsible for circadian regulation ofS-adenosylmethionine decarboxylase gene expression in carnation

S-Adenosylmethionine decarboxylase (SAMDC) activity and mRNA amount increase in a light-dependent manner, and the accumulation ofSAMDC transcripts might be partially regulated by a circadian clock in tritordeum andPharbitis nil. However, no research has been reported on the circadian regulation of SAMDC activity and corresponding gene expression. Here, we determined that SAMDC activity and its mRNA accumulation changed diurnally in carnation leaves. To identify the promoter region responsible for this rhythmicity, we performed northern blot analysis using aGUS cDNA probe in transgenic tobacco plants. The CCS gene was under the control of serial deletions of the 5′-flanking region of a genomic clone corresponding toCSDC9 (gCSDC9). The region between -754 and -274, which negatively regulated the accumulation ofGUS mRNA, had two HD-Zip-2 binding sequences (TAATAATTA) that were found by PLACE analysis of the promoter region. Another region at 273 bp upstream from the transcription initiation site was sufficient for diurnal expression ofGUS, and contained the putative sites responsible for diurnal expression, i.e., the GT-1 consensus sequence (GGTAAT) and a sequence necessary for this circadian expression (CAACTTCATC).