ABSTRACT1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KE-FAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.The simplest olefin, ethylene, is a gaseous plant hormone which regulates many physiological processes of plant growth and development. The biosynthetic pathway for ethylene in higher plants has been elucidated as follows:. Two enzymes that are unique to this pathway are ACC synthase and ACC oxidase (also known as ethylene-forming enzyme). ACC synthase converts S-adenosylmethionine to ACC, and ACC oxidase catalyzes the oxidation of ACC to ethylene. In fruits, both ACC synthase and ACC oxidase are induced during ripening and contribute to the regulation of ethylene biosynthesis.While much progress has been achieved on the characterization of ACC synthase at the biochemical and molecular levels (1-5), progress on the isolation of in vitro ACC oxidase has been slow. ACC oxidase activity is readily demonstrated in vivo by supplying tissues with ACC. These in vivo studies have established that the enzyme has a high affinity for its substrate ACC, exhibits stereospecificity toward the stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid for the synthesis of 1-butene, and specifically converts ACC to ethylene, CO2, and cyanide (6). Although a number of plant enzyme preparations were shown to be capable of oxidizing ACC to ethylene, these preparations lacked these characteristics. Based on these criteria it was concluded that these reported in vitro systems were not the authentic ACC oxidase that functions in vivo (6).Recent advances in the molecular biology of this enzyme have led to the successful isolation ofauthentic ACC oxidase. Based on the observations that ACC oxidase activity in tomato fruit was greatly reduced by an antisense gene of a ripening-related cDNA, pTOM13, Hamilton et al. (7) suggested that the pTOM13 gene was related to ACC oxidase. Later work confirmed that pTOM13 and relat...

Auxin is known to promote ethylene production in vegetative tissues by increasing the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase; therefore, we have studied the effect of auxins on ACC synthase mRNA expression. Total RNA was isolated from auxin-incubated cultured apple (Malus sylvestris Mill.) shoots or mung bean (Vigna radiata L.) hypocotyls. These RNAs and a set of oligonucleotide primers corresponding to two conserved amino acid sequences (SNPLGTT and MSSFGLV) found in ACC synthases isolated from other species were used for polymerase chain reaction-based amplification of DNA fragments encoding the ACC synthase-active site domain. We obtained and sequenced a 290-base pair cDNA fragment (pAA1) from cultured apple shoots and a 328-base pair cDNA clone (pMBAI) from mung bean hypocotyls. Comparisons of their deduced amino acid sequences with those of previously characterized ACC synthase cDNAs indicate that both fragments are, indeed, closely related to ACC synthase cDNA. Northem blot analyses further showed that the expression of these transcripts is regulated by auxin treatment. These data indicate that auxin induces ethylene production transcriptionally by increasing the ACC synthase transcripts. The pAAI shares 46% amino acid sequence homology with ripening-regulated apple fruit ACC synthase, indicating that ripening-regulated and auxin-regulated ACC synthases are encoded by different genes. In mung bean hypocotyls, aminooxyacetic acid, a potent inhibitor of ACC synthase activity, promoted the expression of auxin-induced ACC synthase mRNA, but cycloheximide inhibited this induction.

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)(+) RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

In climacteric fruits, the essential role of ethylene in initiating fruit ripening has been well established. Ethylene is biosynthesized from methionine via S-adenosylmethionine and ACC2 in higher plants (1). ACC oxidase (also known as the ethylene-forming enzyme) catalyzes the oxidation of ACC to ethylene, the final step of this biosynthetic pathway. Unlike vegetative tissues, in which ACC synthase is the rate-limiting enzyme, ripening-associated ethylene production in fruit tissues is regulated by both ACC synthase and ACC oxidase activities (4). To study the molecular regulation of ethylene biosynthesis during apple fruit ripening, it is essential to isolate and characterize the ACC synthase and ACC oxidase genes (Table I). We previously cloned a cDNA encoding apple ACC synthase from apple fruit (2). Two homologous tomato ACC oxidase cDNA clones were isolated and functionally expressed in yeast (3) and Xenopus oocytes (6). In addition, two homologous sequences were isolated from avocado fruit (5) and carnation flowers (7). After comparing the tomato ACC oxidases with the other reported homologous sequences, we constructed two degenerate oligonucleotide primers, corresponding to the conserved sequences of ACENWGF and KFQAKEP (underlined in Fig. 1). A cDNA library made from mRNA isolated from ripe apple fruit was constructed with poly(dA) tailing at the 3' end and poly(dC) tailing at the 5' end (2). PCR-based amplifications were carried out using the apple cDNA library as the template and poly(dC), poly(dT), and the degenerate oligonucleotides as the primers. The PCR products were sequenced, and the overlapping sequences between three PCR products were combined and fused to generate a full-length cDNA designated as pAE 12. The sequence of pAE12 is 1199 base pairs long and contains an open reading frame of 314 amino acids (Fig. 1) homologous to those of tomato ACC oxidase cDNA (3, 6) and other related sequences (5, 7). The sequence homology suggests that the cloned cDNA pAE12 may encode ACC oxidase in apple fruit. This view is supported by our recent observation that the transcript of this gene was absent in preclimacteric fruit but increased dramatically in climacteric fruits (our unpublished results). ACKNOWLEDGMENT We are grateful to Dr. Alan B. Bennett and Mr. Bradford Hall for synthesizing the oligonucleotides.

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The absorption and Raman spectra of hafnium dimers in an argon matrix have been measured. Four weak dimer absorption bands were found between 300 and 700 nm. Resonance Raman spectra (obtained by exciting into a band centered at 620 nm) give w:' = 176.2 (26) cm-l with w a x , < 1 cm-l. Our results represent the first experimental or theoretical study of the dihafnium molecule.

Twenty-seven diterpenes of six chemical classes, including seven new diterpenes (1, 2, 6, 10, 11, 16, and 19), have been isolated from a collection of the brown alga Dictyota plectens from the South China Sea. The structures of the new diterpenes were elucidated by extensive spectroscopic analysis and by comparison with reported data. In the in vitro assays, 9, 12, 14, 16, and 22 showed inhibitory activity against HIV-1 replication with IC50 values of 16.1-30.5 μM, compounds 5, 13, 24, and 26 exhibited anti-H5N1 activity with inhibition rates of 50%-62% at 30.0 μM, and 12 and 24 also showed potent inhibition against LPS-induced NO production with inhibition rates of 90% and 86%, respectively, at 10.0 μM.

Ten new cembrane-based diterpenes, locrassumins A–G (1–7), (–)-laevigatol B (8), (–)-isosarcophine (9), and (–)-7R,8S-dihydroxydeepoxysarcophytoxide (10), were isolated from a South China Sea collection of the soft coral Lobophytum crassum, together with eight known analogues (11–18). The structures of the new compounds were determined by extensive spectroscopic analysis and by comparison with previously reported data. Locrassumin C (3) possesses an unprecedented tetradecahydrobenzo[3,4]cyclobuta[1,2][8]annulene ring system. Compounds 1, 7, 12, 13, and 17 exhibited moderate inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC50 values of 8–24 μM.