Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

Calcium stimulation of ethylene production induced by 1-aminocyclopropane-1-carboxylic acid and indole-3-acetic acid

Journal of Plant Growth Regulation - Ca2+ stimulates 1-aminocyclopropane-1-carboxylic acid (ACC)- and indole-3-acetic acid (IAA)-dependent ethylene production in mung bean hypocotyls and senescing...     

Expression and regulation of the ethylene receptor PpERS gene during pear fruit development and following salicylic acid treatment

A gene encoding an ethylene receptor protein was isolated from pear (Pyrus pyrifolia). This gene, designated PpERS (GenBank accession No. KC517482), was 1,918 bp in length with an open reading frame encoding a protein of 638 amino acids that shared high similarity with another pear ethylene receptor protein PpERS1, and two apple ethylene receptor proteins MdERS and MdERS1. The PpERS was grouped into the ETR1 subfamily of ethylene receptor based on its conserved domain and phylogenetic status. The PpERS gene contained five exons interrupted by four introns. Quantitative RT-PCR indicated that PpERS was differentially expressed in pear tissues and predominantly expressed in petals, shoots, anthers, and 160 days after full bloom fruit. The PpERS expression was regulated during fruit development. In addition, the PpERS gene expression was regulated by salicylic acid (SA) and ethylene in fruit. The results indicated that PpERS might participate in ethylene and SA signaling transduction during pear fruit development.     

Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening

Changes in the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) fruit pericarp tissue during development through ripening were measured by GC-SIM-MS using d₃-ABA and ¹³C₆-IAA internal standards. In the two cultivars of fieldgrown tomatoes analyzed, the highest IAA levels (8–24 ng/g fw) were found at the earliest stage of development (7 days after anthesis) followed by a rapid decline in levels of the hormone. ABA levels of 40–60 ng/g fw were found at the earliest stages of development followed by a decline in levels until ripening occurred when elevated ABA levels (125 ng/g fw) were measured.     

The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

Metabolism of indole-3-acetic acid by orange (Citrus sinensis) flavedo tissue during fruit development

[5-3H, 1'-14C, 13C6, 12C] Indole-3-acetic acid (IAA), was applied to the flavedo (epicarp) of intact orange fruits at different stages of development. After incubation in the dark, at 25 degrees C, the tissue was extracted with MeOH and the partially purified extracts were analyzed by reversed phase HPLC-RC. Six major metabolite peaks were detected and subsequently analyzed by combined HPLC-frit-FAB MS. The metabolite peak 6 contained oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp) and also indole-3-acetyl-N-glutamic acid (IAGlu). The nature of metabolite 5 remains unknown. Metabolites 3 and 4 were diastereomers of oxindole-3-acetyl-N-aspartic acid (OxIAAsp). Metabolite 2 was identified as dioxindole-3-acetic acid and metabolite 1 as a DiOx-IAA linked in position three to a hexose, which is suggested to be 3-(-O-beta-glucosyl) dioxindole-3-acetic acid (DiOxIAGlc). Identification work as well as feeding experiments with the [5-3H]IAA labeled metabolites suggest that IAA is metabolized in flavedo tissue mainly through two pathways, namely IAA-OxIAA-DiOxIAA-DiOxIAGlc and IAA-IAAsp-OxIAAsp. The flavedo of citrus fruit has a high capacity for IAA catabolism until the beginning of fruit senescence, with the major route having DiOxIAGlc as end product. This capacity is operative even at high IAA concentrations and is accelerated by pretreatment with the synthetic auxins 2,4-D, NAA and the gibberellin GA3.     

Tissue localization of a submergence-induced 1-aminocyclopropane-1-carboxylic acid synthase in rice

At least two 1-aminocyclopropane-1-carboxylic acid synthase genes (ACS) are implicated in the submergence response of rice (Oryza sativa). Previously, the OS-ACS5 gene has been shown to be induced during short- as well as long-term complete submergence of seedlings and to be controlled by a balance of gibberellin and abscisic acid in both lowland and deepwater rice. This study demonstrates that OS-ACS5 mRNA is localized in specific tissues and cells both during normal development and in response to complete submergence. The temporal and spatial regulation of OS-ACS5 expression is presented by in situ hybridization and histochemical analysis of beta-glucuronidase (GUS) activity in transgenic rice carrying an OS-ACS5-gus fusion. Whole-mount in situ hybridization revealed that in air-grown rice seedlings, OS-ACS5 was expressed at a low level in the shoot apex, meristems, leaf, and adventitious root primordia, and in vascular tissues of nonelongated stems and leaf sheaths. In response to complete submergence, the expression in vascular bundles of young stems and leaf sheaths was strongly induced. The results of histochemical GUS assays were consistent with those found by whole-mount in situ hybridization. Our findings suggest that OS-ACS5 plays a role in vegetative growth of rice under normal conditions and is also recruited for enhanced growth upon complete submergence. The possible implication of OS-ACS5 in root-shoot communication during submergence stress and its putative role in aerenchyma formation upon low-oxygen stress are discussed.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Selected ion monitoring/isotope dilution mass spectrometric determination of 1-aminocyclopropane-1-carboxylic acid levels in ripening tomato fruit

A new method is described for the quantitation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants. [2,2,3,3,-2H4]ACC has been synthesized and used as an internal standard for selected ion monitoring/isotope dilution quantitation of this compound in ripening tomato fruit. These data are compared with those derived from the widely used indirect oxidative ACC assay (which underestimated the ACC levels by between two- and fourfold). The greater accuracy, sensitivity (100X), and specificity of the mass spectrometric method will be of considerable benefit to those interested in factors which control ACC and ultimately ethylene levels since it is believed that ACC synthesis and its oxidative metabolism to ethylene are the key points at which ethylene biosynthesis is regulated.     

Expression of a single-chain antibody against indole-3-acetic acid in Escherichia coli

A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.     

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out duringadventitious root formation in avocado microcuttings. Both auxinand conjugate were monitored in control cuttings (rooted inthe absence of auxin) as well as in cuttings treated with arooting promotor (IBA) or an auxin transport inhibitor (TIBA).Additionally, a histological study to follow root differentiationwas carried out. In control cuttings IAA levels remained constantthroughout the rooting process, however, in IB A-treated cuttingsIAA levels increased 2-fold during the first 6 d. Addition of200 µM TIBA induced a slight decrease of IAA levels andinhibited root formation. As for IAAsp levels, both control and IBA-treated cuttings showeda big increase before root differentiation occurred and as theprocess went on, a progressive decrease took place. However,in TIBA-treated cuttings IAAsp levels not only did not increasebut diminished progressively during the process. The role ofauxin conjugates during the rooting process of avocado is discussed. Key words: Avocado, IAA, IAAsp, rooting     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

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.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.