ACC Oxidase Transcripts Research Articles

Editing of 1-aminocyclopropane-1-carboxylate oxidase genes negatively affects petunia seed germination.

Editing of ACO genes involved in ethylene biosynthesis pathway reduces ethylene production in petunia seeds and inhibits seed germination. Ethylene production in the seeds of Petunia hybrida cv. 'Mirage Rose' was associated with expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes (PhACO1, PhACO3, and PhACO4). Suppression of their expression by ethylene inhibitor silver thiosulphate (STS) significantly reduced ethylene production and inhibited seed germination. When it was combined with ethylene precursor ACC, ethylene production was re-promoted via activation of the genes and higher seed germination was restored. This was confirmed using the mutants editing the genes and WT. In the present study, compared with wild type plants, three different mutants (phaco1, phaco3, and phaco4) showed significantly decreased germination percentages as well as delayed germination time and seedling growth. These reductions were associated with lighter seed weight, lower ACO transcript levels, and lower ethylene production in mutants. Inhibited seed germination owing to reduced ethylene production was further verified by the supplementation of exogenous ACC and gibberellic acid (GA3) to growth medium, which restored high seed germination activity in all mutants via enhanced ethylene production. In this study, we reported a key regulatory role of ethylene in seed germination mechanisms in petunia. Further, we highlighted on need to consider the negative effects of ethylene reduction in seed germination and plant growth when editing genes in the ethylene biosynthesis pathway for the maintenance of postharvest fruit, vegetable, and flower quality.

Different expression of kiwifruit ethylene-related genes during low storage temperatures

Autocatalytic ethylene production is the start of a transcription cascade that regulates many important features of fruit quality, including flavoring and softening. Kiwifruit (Actinidia deliciosa) is a climacteric fruit, sensitive to low concentrations of ethylene. In current study, the ethylene biosynthesis at low temperatures (− 0.8, − 0.5 and 0 °C) in A. deliciosa has been investigated. The gene expression of transcription factors (AdERF1 and AdETR1) and the enzymes related to ethylene biosynthesis (AdACO1, AdACO2 and AdACS) have been examined using RT-qPCR. According to the results, for all of the examined genes, the highest expression level was observed at − 0.8 and − 0.5 °C, while the lowest expression level was at 0 °C during storage. However, kiwifruit is usually stored at temperatures below 0 °C. Also, the expression of ACC (1-aminocyclopropane-1-carboxylic acid) oxidase genes (AdACO1 and AdACO2) was higher than that of the other genes, because a low storage temperature is responsible for the activation of the ACC synthase transcript at first and later the ACC oxidase (ACO) transcript. Thus, ACO gene induction appears to be the second signal of chilling stress. The induction of ACO transcripts by chilling preceded the appearance of chilling symptoms. According to the current results, the temperature of 0 °C is the best temperature for storing kiwifruit for a long time.

Transient induction of a subset of ethylene biosynthesis genes is potentially involved in regulation of grapevine bud dormancy release.

Transient increases in ethylene biosynthesis, achieved by tight regulation of transcription of specific ACC oxidase and ACC synthase genes, play a role in activation of grapevine bud dormancy release. The molecular mechanisms regulating dormancy release in grapevine buds are as yet unclear. It has been hypothesized that its core involves perturbation of respiration which induces an interplay between ethylene and ABA metabolism that removes repression and allows regrowth. Roles for hypoxia and ABA metabolism in this process have been previously supported. The potential involvement of ethylene biosynthesis in regulation of dormancy release, which has received little attention so far, is now explored. Our results indicate that (1) ethylene biosynthesis is induced by hydrogen cyanamide (HC) and azide (AZ), known artificial stimuli of dormancy release, (2) inhibitors of ethylene biosynthesis and signalling antagonize dormancy release by HC/AZ treatments, (3) ethylene application induces dormancy release, (4) there are two sets of bud-expressed ethylene biosynthesis genes which are differentially regulated, (5) only one set is transiently upregulated by HC/AZ and during the natural dormancy cycle, concomitant with changes in ethylene levels, and (6) levels of ACC oxidase transcripts and ethylene sharply decrease during natural dormancy release, whereas ACC accumulates. Given these results, we propose that transient increases in ethylene biosynthesis prior to dormancy release, achieved primarily by regulation of transcription of specific ACC oxidase genes, play a role in activation of dormancy release.

Pollination of Orchid Flowers: Quantitative and Domain-Specific Analysis of Ethylene Biosynthetic and Hormone-Induced Gene Expression

Pollination initiates a suite of postpollination developmental events in orchid flowers. Ethylene plays a key role in coordinating these developmental changes. We examined the temporal-spatial patterns of expression of genes encoding 1-aminocyclopropane 1-carboxylate acid (ACC) synthase and ACC oxidase following pollination and associated hormonal treatments using quantitative procedures. We present the first in situ hybridization analysis of ACC synthase and an earlier time course of ACC oxidase gene expression. In immature ovaries, critical morphological changes involving cell differentiation and growth were affected by pollination and various hormone and inhibitor treatments. Patterns of ACC synthase and ACC oxidase mRNA accumulation were determined by RNA blot hybridization analysis. Gene expression analysis using real-time quantitative polymerase chain reaction indicated that the unpollinated ovary had very low levels of ACC oxidase expression at 6 h and that pollination caused near-maximal levels in this organ by 12 h. Quantitative mRNA analysis showed that auxin could mimic pollination by inducing a similar pattern of organ-specific expression, but earlier and to higher levels. Ethylene had the most dramatic effect by inducing gene activity to its highest levels at all time points and in all floral organs. The early induction in situ hybridization analyses contribute new findings that ACC synthase and ACC oxidase transcripts were localized sequentially in epidermal, phloem, and parenchyma cells of the stigma and that by 8–12 h postpollination, saturation had occurred throughout the stigma. Finally, phylogenetic analysis of orchid ACC synthase proteins leads to the conclusion that at least a subgroup of the angiosperm ACC synthase gene family is induced by auxin but may not be posttranscriptionally regulated, indicating regulatory sequence but not functional divergence of orchid genes with other seed plant ACC synthase proteins.

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit.

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions.

UV-C radiation modifies the ripening and accumulation of ethylene response factor (ERF) transcripts in tomato fruit

Ultraviolet-C (UV-C) radiation is used as a postharvest treatment to prolong the shelf life of fruit. However, this stressful process may also affect ethylene production and, consequently, the expression of genes encoding ethylene response factors (ERFs). To test this hypothesis, MicroTom tomatoes harvested at the breaker stage were subjected to: 1 – application of 3.7kJm−2 UV-C radiation, 2 – application of 2μLL−1 1-methylcyclopropene (1-MCP) followed by UV-C radiation; and 3 – without 1-MCP or UV-C (control treatment). After treatment all fruit were stored for 12d at 21±2°C and 80±5% relative humidity (RH). Although UV-C radiation increased ACC oxidase transcripts and stimulated ethylene production, the ripening evolution was delayed. Fruit treated with UV-C showed lower accumulation of lycopene, β-carotene, lutein+zeaxanthin and δ-tocopherol; but retained higher levels of chlorogenic acid, ρ-coumaric acid and quercetin after 6d. Additionally, UV-C treated fruit had higher contents of polyamines (putrescine and spermidine). Among the 14 ERFs studied, 11 (Sl-ERF A.1, Sl-ERF A.3, Sl-ERF B.1, Sl-ERF B.2, Sl-ERF B.3, Sl-ERF C.6, Sl-ERF D.1, Sl-ERF D.3, Sl-ERF E.1, Sl-ERF F.5, Sl-ERF G.2) exhibited increased transcript accumulation, 2 ERFs (Sl-ERF E.2 and Sl-ERF E.4) showed decreased transcript accumulation and only 1 ERF (Sl-ERF E.3) was not significantly affected by UV-C treatment. As expected, the transcript profiles of 1-MCP and/or UV-C-treated tomatoes demonstrate that ethylene plays an important role in the expression of ERFs. The delay in fruit ripening may be caused by the activation of ERFs that could act as regulators of metabolic pathways during ripening. However, this hypothesis needs to be better tested. In conclusion, a relationship has been established between UV-C treatment and ripening delay, correlated to changes in 13 ERF transcripts evaluated during postharvest treatment.

Elevated H2O2 production via overexpression of a chloroplastic Cu/ZnSOD gene of lily (Lilium oriental hybrid ‘Marco Polo’) triggers ethylene synthesis in transgenic potato

Transgenic potato plants (SS2 and SS4) that overexpressed a chloroplastic copper/zinc superoxide dismutase lily gene were utilized as an H(2)O(2)-inducible system in order to study the role of H(2)O(2) as a signaling molecule in the biosynthesis of ethylene. SS2 and SS4 plants grown in vitro under sealed microenvironment (SME) conditions displayed anomalous phenotypes including reduction of stem elongation, radial stem growth, and promotion of root hair formation in the generated root, which were similar to ethylene-induced responses. In addition, SS4 plants showed severe vitrification in developing leaves and elevated ethylene production under SME conditions. After the ethylene action inhibitor AgNO(3), 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) inhibitor CoCl(2), and ACC synthase inhibitor L -aminoethoxyvinylglycine were added to the growth media, the anomalous phenotypes in SS4 plants reverted to their normal phenotype with a concurrent decrease in ethylene production. Northern blot analysis showed that ACO transcripts in SS4 plants were constantly at high levels under normal and SME conditions, indicating that a high level of H(2)O(2) in SS4 plants up-regulates ACO transcripts. Moreover, the direct treatment of H(2)O(2) in potato plants confirmed the elevated expression of the ACO gene. Taken together, these data suggest that the high concentration of H(2)O(2) in transgenic potato plants stimulates ethylene biosynthesis by activating ACO gene expression.

Suppression of the expression of genes encoding ethylene biosynthetic enzymes in harvested broccoli with high temperature treatment

Abstract Broccoli ( Brassica oleracea L. var. Italica) senesces very rapidly at room temperature after harvest. Postharvest high temperature treatment reduced ethylene production and delayed yellowing of florets during storage. High temperature treatment suppressed 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity and the accumulation of ACS transcripts ( BO-ACS1 ). Increase in activity was correlated with that of ethylene production. Changes in ACC synthase activity seemed to reflect the expression of the BO-ACS1 gene with a 1 day lag. High temperature treatment also suppressed ACC oxidase (ACO) activity. However, the changes did not seem to be related to those in ethylene production. High temperature treatment seemed to affect the accumulation of ACO transcripts ( BO-ACO1 and BO-ACO2 ) differently; the accumulation of BO-ACO1 transcripts was delayed and that of BO-ACO2 decreased. These results suggest that inhibition of ethylene production with high temperature treatment could be due to suppression of BO-ACS1 gene expression.

Effect of p-CPA-parthenocarpic setting on the delayed ripening of netted-melon fruits

Application of plant growth regulators may induce parthenocarpic fruit setting, avoiding problems due to poor pollination. Their influence on the ripening of netted-melon fruit (Cucumis melo L.), however, is not yet well understood. The ripening behavior of parthenocarpic melon fruit (treated with 50 ppm 4-chlorophenoxy acetic acid (p-CPA) and 350 ppm gibberellic acid (GA3) at anthesis) was compared with that of hand-pollinated fruit. The ethylene production in parthenocarpic fruit peaked up about 10 days behind that of pollinated fruit but was greater. The delay was accompanied by a retardation in fruit softening, thereby leading to an extended edible period. The expression of ethylene synthesis-related enzymes: ACC synthase (ACS) and ACC oxidase (ACO) genes at the commencement of the ripening process was also evaluated. Northern blot and RT-PCR analyses revealed a higher level in the mRNA transcript abundance of ACS genes (auxin- and wound-inducible) but lower abundance of the ripening-related gene ACO in parthenocarpic fruit compared to that of pollinated fruit. Western blot analysis further confirmed the lower level of ACO polypeptide in parthenocarpic fruit. The retarded ripening of parthenocarpic melon fruit resulted from the delayed production of ethylene, which is likely a consequence of the low abundance of ACO. A possible inhibitory effect of auxin on ACO transcript as a result of p-CPA application is proposed.

Effects of salinity, high irradiance, ozone, and ethylene on mode of photosynthesis, oxidative stress and oxidative damage in the C3/CAM intermediate plant Mesembryanthemum crystallinum L.

ABSTRACTThe occurrence of oxidative stress and oxidative damage in the halophyte Mesembryanthemum crystallinum during C3/crassulacean acid metabolism (CAM) transition evoked by salinity and high irradiance was investigated. The hypothesis tested was that CAM induction will effectively protect M. crystallinum against oxidative damage even under additionally applied oxidative stress via ozone (150 p.p.b.) exposure. Salinity (0.4 m NaCl), high irradiance and ozone induced the cytosolic CuZn‐superoxide dismutase (SOD; EC 1.15.1.1) transcript abundance and activity indicating oxidative stress. However, ozone did not induce CAM. Contrary to the hypothesis, that ethylene may be involved in CAM induction, gassing with ethylene (200 p.p.b.) did not lead to CAM, but revealed that in M. crystallinum ethylene biosynthesis is induced by autostimulation. A putative fragment of the hitherto unknown nu‐cleotide sequence of 1‐aminocyclopropane‐1‐carboxylate oxidase (ACC; EC 4.4.1.14) was isolated and used as a specific probe against the ACC oxidase transcript to monitor oxidative damage. Ozone exposure increased the ACC oxidase expression only in plants with C3‐photosynthesis which also showed severe necrosis and reduced photosynthetic activity, whereas plants in CAM mode (salt induced) remained unaffected. Natural stressors such as salinity and high irradiance induce CAM and up‐regulate the ARS component CuZn‐SOD very effectively to protect M. crystallinum from oxidative damage even when additional oxidative stress such as ozone is present.

Expression and activities of ethylene biosynthesis enzymes during ripening of banana fruits and effect of 1-MCP treatment

The respiratory climacteric, ethylene evolution and activities and expression of ethylene biosynthesis enzymes show a characteristic pattern in banana during ethylene induced ripening. A unique biphasic respiratory climacteric with a 10 and 6 fold increment in respiration rates on days 2 and 6 respectively after ethylene treatment is preceded by ethylene evolution on days 1 and 4 with 8.3 and 6.93 fold increments respectively. This represents a unique feature of ripening in banana. While ACC synthase transcript accumulation matched the respiratory climacteric, the ACC synthase activity and ACC oxidase transcript accumulation showed a different pattern. The most significant observation was the effect of 1-MCP on ACC content and in vitro ACC oxidase activity. Though 1-MCP treated fruit did not show any respiratory climacteric or burst in ethylene production, it did not inhibit completely ACC accumulation and in vitro ACC oxidase activity. No transcript accumulation of ACC synthase was observed at any time in 1-MCP treated fruits, whereas a basal level of ACC oxidase transcript was detected throughout. It is concluded that ethylene induced ripening of banana is characteristically different from that of other climacteric fruits and that ethylene biosynthesis may have more than one mechanisms operating during ripening which are tightly controlled at various levels.

Dynamic 1-aminocyclopropane-1-carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles.

In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes.

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.

Induction of defense responses in tobacco by the protein Nep1 from Fusarium oxysporum

Fusarium oxysporum produces a 24-kDa protein, Nep1, which induces necrosis and ethylene production in leaves of many dicot plant species. Detached Nicotiana tabacum L. cv. Xanthi leaves respond with concentration-dependent necrosis after infiltration with Nep1 or when Nep1 is taken up by the vascular tissue. This response follows the induction of ethylene biosynthesis and accumulation of ACC synthase and ACC oxidase transcripts. Pretreating the leaves with 100 μl/l ethylene prior to elicitation enhanced Nep1-induced ethylene production. Nep1 (208 nM) causes extensive necrosis of mature tobacco leaf tissue when applied to Xanthi tobacco as a foliar spray (129 ml/m 2). Tobacco cell cultures respond to Nep1 by alkalization of the culture media, the accumulation of potassium in the media, oxygen uptake, induction of active oxygen species, and eventual cell death. The response of cultured tobacco cells to Nep1 is time- and concentration-dependent. Cell death was the same at 300 min for 5 ng/ml and higher concentrations, while 0.5 ng/ml had no effect on cell death. In the case of O 2 uptake, cells responded to 0.5 ng/ml within minutes of treatment, but at a rate lower than 5 ng/ml. The lower concentration of Nep1 did not induce an increase in pH, K + efflux, or increasing H 2O 2 accumulation in the culture media.

Expression of genes for ethylene biosynthetic enzymes and anethylene receptor in senescing flowers of miniature potted roses

SummaryExpression of genes for the ethylene biosynthetic enzymes ACC oxidase and ACC synthase, and the gene for a putative ethylene receptor, termed RhETR, in miniature roses (Rosa hybrida) were examined during flower senescence of the long-lasting cv. Vanilla and cv. Bronze, which has a short flower life. The abundance of ACC oxidase transcript increased during the late stages of flower development in petals of both cultivars, being higher for `Bronze' than for `Vanilla'. The ACC synthase transcript increased during flower senescence in `Vanilla' but remained constant at a low level in `Bronze'. The expression of RhETR was distinctly higher in the cultivar with short flower life (`Bronze') than in the long-lasting cultivar (`Vanilla'), and modulation of receptor levels was also observed during flower development. Exposure to low ethylene concentrations resulted in an up-regulation of RhETR in flowers of both cultivars. Differences in the expression of the putative ethylene receptor of cultivars with short or long flower life suggest that variation in flower longevity may be due to differences in receptor levels during flower development.

The physiological and molecular responses of carnation flowers to high temperature

Abstract Carnation ( Dianthus caryophyllus cv. `White Sim') flowers were subjected to a heat treatment to investigate the physiological and molecular effects of high temperatures on flower senescence. Flowers were exposed to 44°C for 24 h in the dark. Control flowers were held at 23°C. In heat-treated flowers the ethylene climacteric occurred 120 h after treatment, a delay of 24 h when compared to control flowers. Maximum ethylene production was decreased from 44 to 31 nl g −1 h −1 in heat-treated flowers. Northern blot analysis of the ethylene biosynthetic genes ACC synthase and ACC oxidase indicated that the accumulation of these mRNAs is delayed by 24 h in heat-treated flowers. The accumulation of senescence-related (SR) genes followed a similar pattern. Further investigation revealed a decreased responsiveness to exogenous ethylene and a reduced capacity to produce ethylene in petals from heat-treated flowers. Northern blot analysis again revealed a delay in the accumulation of ACC synthase and ACC oxidase transcripts. SR gene expression induced by ethylene, however, was not affected by the heat treatment. The beneficial effects of high temperatures, a delay in ethylene production and reduced responsiveness to ethylene, may lead to horticultural application.

Use of transgenic plants to study the role of ethylene and polygalacturonase during infection of tomato fruit by Colletotrichum gloeosporioides

The infection of tomato fruit by the postharvest pathogen Colletotrichum gloeosporioides did not proceed until the onset of ripening in response to ethylene. Compared with fruit from wild‐type plants, infection progressed more slowly in transgenic fruit in which ethylene biosynthesis and ripening had been inhibited by an ACC oxidase (ACO) antisense transgene. In contrast, transgenic fruit deficient in polygalacturonase developed lesions at the same rate as the wild‐type fruit. Ethylene biosynthesis increased rapidly in response to infection of ripe wild‐type and ACO antisense fruit but was 25 times greater in the former. Fruit from the ripening mutant ripening inhibitor (rin), which are normally very resistant to infection, became infected quickly when incubated in the presence of ethylene, whereas fruit incubated in the absence of ethylene remained healthy. The ACO 1 mRNA accumulated to detectable levels within 24 h of inoculation of unripe wild‐type fruit, prior to the development of visible symptoms, whereas there was no detectable expression in the inoculated ACO antisense fruit. ACO transcripts accumulated to maximum levels during the early stages of infection of ripe fruit, correlating with maximum ethylene biosynthesis. Northern analysis using gene‐specific probes for each of the three ACO genes indicated that ACO 1 was the main gene expressed in response to infection and that there was no detectable expression of ACO 2 and 3.

Reduction in ethylene synthesis in parthenocarpic Actinidia deliciosa fruit induced by N-(2-chloro-4-pyridyl)-N′-phenylurea

Parthenocarpic kiwifruit Actinidia deliciosa was produced and induction of ethylene synthesis in response to ethylene treatment studied. At anthesis, flowers were treated with N -(2-chloro-4-pyridyl)- N ′-phenylurea (CPPU). CPPU-treated parthenocarpic fruit and pollinated normal fruit were harvested in early November, and then treated with 1000 μ l l −1 ethylene at 20°C for 24 h. Ethylene production following ethylene treatment was reduced and delayed in the parthenocarpic fruit. To investigate the mechanism of this reduction in ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations, and gene expression of ACC synthase (using a cDNA probe, AD-ACS1) and of ACC oxidase (with a cDNA probe, AD-ACO1) were analyzed. ACC concentrations and levels in ACC synthase transcripts increased in the pollinated fruit 2 days after the 24 h ethylene treatment, but not in the parthenocarpic fruit. Accumulation of ACC oxidase transcripts was stimulated immediately by the 24 h ethylene treatment in both pollinated and parthenocarpic fruit. These results suggest that suppression of ethylene synthesis in parthenocarpic fruit resulted mainly from suppressed expression of ACC synthase.

Ethylene biosynthesis in banana fruit: isolation of a genomic clone to ACC oxidase and expression studies

In this paper we present the expression studies and structural analysis of one genomic sequence isolated from banana. Using a RNA-based PCR amplification of ACC oxidase cDNAs from ripe banana, we obtained a product of 800 bp which after sequence analysis was found to code for a protein highly homologous to ACC oxidase proteins. This PCR product was used as probe for screening a genomic library and three different groups of clones were obtained as indicated by restriction mapping. One clone (ACO1) was selected for further study and fully sequenced. Comparison of this sequence with the PCR products and other cloned ACC oxidase genes, revealed that ACO1 encoded the transcript in four exons interrupted by three introns. Southern blot analysis showed a major band hybridizing to the PCR probe suggesting that the ACC oxidase gene is present in a single copy or that the genes are clustered as is the case in tomato and carnation. By northern blot analysis it was found that the ACC oxidase transcript appears in the pulp earlier than in the peel but that exogenous ethylene alters this pattern and the transcript is expressed in the peel first.

Induction of 1‐aminocyclopropane‐1‐carboxylate oxidase mRNA by ethylene in mung bean hypocotyls: involvement of both protein phosphorylation and dephosphorylation in ethylene signaling

SummaryEthylene induced an increase in the level of 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase mRNA but suppressed the expression of ACC synthase transcript in mung bean hypocotyls. Induction of ACC oxidase transcript by ethylene was saturated at a low concentration (1–3 µl l−1) and detectable within 1 h after ethylene treatment; 2,5‐norbornadiene, an inhibitor of ethylene action, significantly reduced the basal level of ACC oxidase transcript present in intact mung bean hypocotyls. Treatment with the protein synthesis inhibitor cycloheximide completely inhibited the ethylene‐induced accumulation of ACC oxidase transcript, indicating that de novo protein synthesis is necessary for expression of the ethylene‐inducible ACC oxidase gene. ABA decreased the ethylene‐induced accumulation of ACC oxidase mRNA, but restored the ethylene‐suppressed level of ACC synthase (VR‐ACS1) transcript. The protein kinase inhibitor staurosporine effectively prevented ethylene‐induced ACC oxidase gene expression, whereas it substantially recovered the ethylene‐suppressed transcript level of ACC synthase, suggesting that protein phosphorylation plays a role in the induction of ACC oxidase and the suppression of ACC synthase by ethylene in mung bean hypocotyls. Okadaic acid, a potent inhibitor of protein phosphatase, did not affect the expression of ACC oxidase. However, addition of okadaic acid along with ethylene effectively blocked the ethylene‐induced accumulation of ACC oxidase transcript, while there was an increase in the level of ethylene‐suppressed ACC synthase transcript. These results indicate that protein dephosphorylation in addition to phosphorylation is necessary for the ethylene signaling that regulates the expression of these genes.