Hiroshi Hyodo

Wound-induced ethylene synthesis in stem tissue of harvested broccoli and its effect on senescence and ethylene synthesis in broccoli florets

Abstract Broccoli ( Brassica oleracea , var. italica ) florets (flower buds) senesced rapidly after harvest at ambient temperatures. ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) was induced rapidly in the cut surface of stem tissue in the first 2 mm thick and the basal portions of curds soon after harvest, leading to an accumulation of ACC. The abundance of one ACS mRNA (BO-ACS1) increased in the first layer, the inner layer of stem (2–4 mm, second layer), and the basal portion of curds, while the transcripts for another (BO-ACS2) increased only in the first layer. Conjugated ACC (malonyl ACC, MACC) levels increased in all portions as senescence progressed. In florets, ACC synthase activity and BO-ACS1 transcripts were detected with no significant changes observed during senescence. ACC levels in florets stayed low throughout the experimental period, whereas MACC levels were much higher than those of ACC. The marked rise in ACC oxidase (ACO) activity in florets was detected almost in parallel with a significant increase in ethylene production. The abundance of ACO transcripts (BO-ACO1 and BO-ACO2) increased concurrently with the rise in ACC oxidase activity. These findings suggest that ACC and ethylene synthesized in the stem in response to wounding may have involved the enhanced activity of ACC oxidase and increased abundance of its transcripts in florets.

Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant proteins

Two distinct clones having high nucleotide identity to the sequences encoding ascorbate peroxidase (APX) were isolated from broccoli (Brassica oleracea L. var. italica). Deduced amino acid sequences of both cDNAs, BO-APX 1 (accession number AB078599) and BO-APX 2 (accession number AB078600), shared identity of 92.8% and there was more than 80% identity between BO-APXs and other plant cytosolic APXs at the protein level. Gene expression and protein levels of BO-APX 1 and BO-APX 2 were investigated in various parts of broccoli after harvest. Transcript levels of BO-APX 2 gradually increased in florets, while those of BO-APX 1 decreased in florets after harvest. BO-APX 1 and BO-APX 2 were expressed in Escherichiacoli as a fusion protein with glutathione S-transferase (GST) and purified to homogeneity by glutathione sepharose 4B column chromatography. Both proteins of BO-APX 1 and BO-APX 2 appeared as a single major band on SDS-PAGE corresponding to a mass of 25 kDa and reacted with polyclonal antibodies raised against recombinant BO-APX 1. Both enzymes showed high specificities for ascorbate and hydrogen peroxide. The km values of recombinant BO-APX1 and BO-APX 2 for ascorbate were 395 and 526 μM and those for hydrogen peroxide were 15 and 7 μM, respectively. The role of APX was discussed in relation to ascorbate breakdown in broccoli florets during senescence.

Wound-induced ethylene synthesis and its involvement in enzyme induction in mesocarp tissue of Cucurbita maxima

Abstract The activity of peroxidase in mesocarp tissue of Cucurbita maxima was rapidly increased after wounding. The increase in peroxidase activity was preceded by increased ethylene production. The induction of peroxidase activity was strongly suppressed by the application of 2,5-norbornadiene (NBD) and aminoethoxyvinylglycine (AVG) to excised discs, indicating that endogenous ethylene produced in response to wounding may have promoted the induction of peroxidase. Raised activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and phenylalanine ammonia-lyase (PAL) were noted prior to increased peroxidase activity. Although no significant increases in soluble phenolics, such as chlorogenic acid, were found in the wounded mesocarp tissue, lignin formation was clearly enhanced by wounding along with the increased level of ethylene production and the enhanced levels of PAL and peroxidase activities. ACC synthase and PAL induced by wounding were observed to be located predominantly in the upper and adjacent 0.5 mm of the cut surface, while peroxidase activity was found to exist deep inside the tissue.

Ethylene biosynthesis in sweet potato root tissue infected by black rot fungus (Ceratocystis fimbriata)

Abstract The rate of ethylene production in sweet potato ( Ipomoea batatas Lam) root tissue greatly increased in response to infection by black rot fungus ( Ceratocystis fimbriata Ell. & Halst.). Ethylene evolved rapidly in the first layer of root cells (0–0.5 mm) 24 h after inoculation with the endoconidia of the fungus. In this layer, the host-parasite interactions were very strong. Ethylene production rate reached a peak (up to 300 nl g −1 h −1 ) 24 h after inoculation, and then declined to a low level. This was followed by an increase in ethylene production in the second layer of cells (0.5–1.0 mm) following continuing invasion by the fungus. Incorporation of l -[ 14 C(U)]methionine into ethylene 24 h after inoculation occurred at a lower rate in the first cell layer than in the second, where the ethylene production rate was about 15-fold less. When ACC (1-aminocyclopropane-1-carboxylic acid) was supplied externally, the rate of ethylene production in the first layer was not enhanced, and neither was [ 14 C]methionine incorporation into ethylene diluted. The activity of ACC oxidase extracted from the first layer was extremely low (less than 10 nl g −1 h −1 ) when compared with the ethylene production rate, although ACC oxidase protein was weakly detected by Western blot analysis in the extract from the infected tissue using antibodies raised against ACC oxidase purified from recombinant Escherichia coli . These results indicate that the predominant ethylene generated in the site adjacent to the invaded region of sweet potato root tissue may originate from a pathway independent of ACC. However, it is possible that the methionine-ACC pathway may operate in tissue internal to the above active site.

Wound-Induced Ethylene Production and 1-Aminocyclopropane-1-Carboxylic Acid Synthase in Mesocarp Tissue of Cucurbita Maxima

ACC synthase activity was rapidly induced in mesocarp tissue of winter squash fruit by wounding, followed by increases in ACC level and in rate of ethylene synthesis. PAL activity was also induced with a similar pattern in the wounded tissue. Induction of both ACC synthase and PAL occurred almost within the surface layer of 1 mm in thickness consisting of about 11 cell layers. Treatment of excised tissue with water prior to incubation largely reduced the subsequent induction of both enzymes, which was partially relieved by the administration of N-acetyl-chitohexaose. Induction of ACC synthase was suppressed by ethylene, while PAL induction was not affected by ethylene exogenously applied to wounded mesocarp tissue.

Ethylene Biosynthesis in sweet-potato Root Tissue Induced by Infection with Black Rot Fungus (Ceratoystis fimbriata)

Ethylene production rapidly increased in sweet potato (Ipomoea batatas Lam.) root tissue in response to infection by black rot fungus (Ceratocystis fimbriata Ell. & Halst). Ethylene production increased in the surface layer (0-0.5mm) of the root inoculated x4ith endoconidia of C fimbriata, reaching a maximum 1 day after inoculation and declining thereafter The rise in the surface layer was followed by a subsequent increase in the inner, second (0.5-1.0mm). and third (1.0-1.5mm) layers as the fungus penetration progressed Discs 10mm in diameter and 0.5mm thick excised from the surface of the infected root slices were used for the experiments Our previous study demonstrated that ethylene synthesis in the infected tissue may occur via a pathway independent of the methionine ACC (1-aminocyclopropane-1-carboxylic acid) pathway Ethylene synthesis in the infected tissue was significantly suppressed by the inhibitors that interfere with NADPH oxidase. phospholipase A2 and lipoxygenase, by metal ion chelators, and by scavengers of reactive oxygen species The inhibition of metal ions was restored by the addition of cupric ion These results suggest that in the infected sweet potato root tissue, polyunsaturated fatty acids are released from membrane phospholipids followed by peroxidation by lipoxygenase The hydroperoxides could be decomposed in the presence of copper ion or copper enzyme and reactive oxygen species such as hydroxyl radical, leading to ethylene generation Dc novo protein synthesis was required for ethylene synthesis to be induced in response to the fungal infection

Biochemical characterization and expression of recombinant ACC oxidase in Escherichia coli and endogenous ACC oxidase from kiwifruit

Abstract To use recombinant 1-aminocyclopropane-1-carboxylate (ACC) oxidase for research on ethylene biosynthesis, the biochemistry of both recombinant and endogenous ACC oxidase from kiwifruit was compared. When induced by the addition of isopropyl-β- d -thiogalactopyranoside (IPTG) to Escherichia coli (E. coli) cells transformed with cDNA AD-ACO1 using the pGEX-4T-1 vector, ACC oxidase identical to that from kiwifruit was expressed as a polypeptide of 37 kDa. Apparent Km values for ACC for both recombinant and endogenous ACC oxidase were 41 and 16 μM, respectively. Both forms of ACC oxidase exhibited absolute requirements for ferrous iron, ascorbate and bicarbonate for maximum activity The activities of both enzymes were inhibited by ethylenediaminetetraacetic acid (EDTA), 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), o-phenanthroline (PA), α-aminoisobutyric acid (AIB), and p-chloromercuriphenylsulfonic acid (PCMPS). Addition of dithiothreitol (DTT) stimulated activity of both ACC oxidases. The results indicate that the recombinant ACC oxidase was similar biochemically to the endogenous kiwifruit enzyme. Western blot analysis using antibody raised against purified transformed ACC oxidase protein showed differential expression of endogenous ACC oxidase protein in kiwifruit during ripening. Expression may start in the columella region and then increase in surrounding tissues with progressive stages of ripening.

Ethylene Production by the Kudzu Strains of Pseudomonas Syringae Pv. Phaseolicola

Significant amounts of ethylene were produced by Pseudomonas solanacearum (all strains), P. syringae pv. phaseolicola (Kudzu strains isolated from Pueraria lobata) and Erwinia rhapontici (2 strains out of 22) out of 24 species, 3 subspecies and 38 pathovars of plant pathogenic bacteria tested in yeast extract-peptone broth. The bean strains of P. syringae pv. phaseolicola causing halo blight in kidney bean plants did not produce ethylene. The Kudzu strains produced ethylene at a rate of 7 to 100x109nl cell-1h-1 which was 500 to 1,000 times higher than that of P. solanacearum and several times higher than that of Penicillium digitatum, the most potent ethylene producer known among microorganisms.