Basal Ethylene Research Articles

WRKY29 transcription factor regulates ethylene biosynthesis and response in arabidopsis

The gaseous phytohormone ethylene participates in a lot of physiological processes in plants. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and the ACC oxidase (ACO, EC 1.14.17.4) are key enzymes in ethylene biosynthesis. However, how ACSs and ACOs are regulated at the transcriptional level is largely unknown. In the present study, we showed that an Arabidopsis (Arabidopsis thaliana) WRKY-type transcription factor (TF), WRKY29 positively regulated the expression of ACS5, ACS6, ACS8, ACS11 and ACO5 genes and thus promoted basal ethylene production. WRKY29 protein was localized in nuclei and was a transcriptional activator. Overexpression of WRKY29 caused pleiotropic effect on plant growth, development and showed obvious response even without ACC treatment. Inducible overexpression of WRKY29 also reduced primary root elongation and lateral root growth. A triple response assay of overexpression and mutant seedlings of WRKY29 showed that overexpression seedlings had shorter hypocotyls than the transgenic GFP (Green Fluorescence Protein) control, while mutants had no difference from wild-type. A qRT-PCR assay demonstrated that expression of multiple ACSs and ACO5 was up-regulated in WRKY29 overexpression plants. A transactivation assay through dual luciferase reporter system confirmed the regulation of promoters of ACS5, ACS6, ACS8, ACS11 and ACO5 by WRKY29. Both in vivo chromatin immunoprecipitation (ChIP)- quantitative PCR and in vitro electrophoretic mobility shift assay (EMSA) revealed that WRKY29 directly bound to the promoter regions of its target genes. Taken together, these results suggest that WRKY29 is a novel TF positively regulating ethylene production by modulating the expression of ACS and ACO genes.

Effect of Ethylene-Insensitive Mutation etr2b on Postharvest Chilling Injury in Zucchini Fruit

Zucchini is a vegetable fruit that is very susceptible to postharvest chilling injury, and fruit ethylene production is correlated with chilling injury sensitivity, such that the more tolerant the cultivar, the lower is its ethylene production. It is expected that zucchini fruit with reduced sensitivity to ethylene would have a higher chilling injury tolerance. In this study, we compared the postharvest fruit quality of wild type and ethylene-insensitive mutant etr2b, in which a mutation was identified in the coding region of the ethylene receptor gene CpETR2B. Flowers from homozygous WT (wt/wt), mutant plants in homozygous (etr2b/etr2b) and heterozygous (wt/etr2b) were hand-pollinated, and all fruits were harvested with the same length, at about 8 days after pollination. After harvesting, fruit of each genotype was randomly divided in 3 batches of 12 fruits each (four replications with three fruits each), and then stored at 4 °C and 95% RH. At 0, 7, and 14 days after cold storage, each batch was used to assess ethylene production, respiration rate, weight and firmness loss, chilling injury, and oxidative stress metabolites. The results showed a lower chilling injury associated with lower cold-induced ethylene production in the mutant fruit, in comparison with the WT fruit. These data demonstrated that the ethylene-insensitive etr2b mutant fruit was more tolerant to chilling injury, confirming that basal ethylene in the still undamaged fruit could function as a modulator of post-harvest chilling injury. Moreover, the higher chilling tolerance of the etr2b mutant fruit was not associated with MDA content, but was concomitant with a reduction in the accumulation of hydrogen peroxide in the refrigerated mutant fruit.

Dendrobium orchids carrying antisense ACC oxidase: small changes in flower morphology and a delay of bud abortion, flower senescence, and abscission of flowers.

Four Dendrobium Sonia 'Earsakul' lines were generated by insertion of one, two or three antisense copies of a Carica papaya gene encoding 1-aminocyclopropane-1-carboxylic acid oxidase (CpACO). Whole vegetative plants of the transgenic lines showed about 50% of the basal ethylene production rate, while the increase in ethylene production in floral buds during opening and open flowers prior to visible senescence was delayed. Detailed analysis of more than 100 parameters in flowering plants showed no effect of antisense ACO on plant morphology and coloration, except for shorter length and width of some of the sepals and petals. In intact plants the water-soaking of floral buds as well as bud abscission were delayed by ACO antisense, as was the time to senescence of open flowers. Pollen viability and pollen tube growth were not affected in the transgenic lines. In cut inflorescences placed in water, bud yellowing, bud water soaking, and bud abscission were considerably delayed by the antisense construct, while the life span of open flowers were increased and abscission of open flowers were delayed. It is concluded that the reduction of ACO activity affected the shape of some petals/sepals and delayed the abortion in floral buds, and the senescence and abscission of open flowers.

Transcriptome analysis reveals interplay between hormones, ROS metabolism and cell wall biosynthesis for drought-induced root growth in wheat

The ability of roots to grow under drought stress is an adaptive trait for crop plants especially under rain fed and restricted irrigation regime. To unravel the molecular mechanism of drought induced-root growth, root transcriptomes of two wheat genotypes viz. Raj3765 and HD2329, with contrasting root growth under drought stress were analyzed. Drought stress significantly enhanced total root length in Raj3765 as compared to that of HD2329. RNA-seq analysis led to the identification of 2783 and 2638 differentially expressed genes (DEGs) in Raj3765 and HD2329, respectively, under drought stress as compared with non-stress conditions. Functional annotation, gene ontology and MapMan analysis of the DEGs revealed differential regulation of genes for pathways associated with root growth and stress tolerance. Drought stress significantly upregulated auxin receptor (AFB2) and ABA responsive transcription factors (MYB78, WRKY18 and GBF3) in roots of Raj3765. Although certain genes for ethylene pathway were downregulated in both the genotypes, ACC oxidase and 2OG-Fe(II) oxygenase were upregulated only in Raj3765 which might contribute to maintenance of a basal ethylene level to maintain root growth. Several genes related to cell wall biosynthesis and ROS metabolism were significantly upregulated in Raj3765. Genes related to gibberellic acid, jasmonic acid and phenylpropanoid pathways were downregulated in roots of both the genotypes under drought stress. Our analysis suggests that a coordinated yet complex interplay between hormones, cellular tolerance, cell wall synthesis and ROS metabolism are required for drought induced root growth in wheat.

Cross-talk modulation between ABA and ethylene by transcription factor SlZFP2 during fruit development and ripening in tomato

The stress hormone ABA not only regulates stress response, but is also required for plant development and growth. Some evidences indicate that ABA plays a pivotal role in the ripening process of non climacteric as well as climacteric fruits. In a recent study, we showed that the tomato (Solanum lycopersicum) transcription factor SlZFP2 fine tunes ABA biosynthesis during fruit development through direct suppression of ABA biosynthetic genes and it also regulates fruit ripening through transcriptional suppression of the ripening regulator CNR. This indicates that SlZFP2 likely modulates the cross-talk between ABA and ethylene in regulation of fruit development and ripening in tomato. Gene expression analysis using ABA deficient mutants sit and flc as well as the SlZFP2 RNAi lines of high fruit ABA production showed that ethylene biosynthetic genes LeACS1A, LeACS1 and LeACO1 were positively regulated by ABA during early fruit growth. We reason that ABA promotes basal ethylene biosynthesis in system 1 during fruit growth and likely plays a minor role in ripening regulation after the onset of ripening process.

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.

Expression of Ethylene Biosynthesis and Signaling Genes during Differential Abscission Responses of Sweet Orange Leaves and Mature Fruit

When applying abscission agents to tree fruit to facilitate harvest, it is desirable to loosen fruit and not leaves or other organs, but mechanisms controlling leaf and fruit drop are not fully understood. The effect of 450 μL·L−1 ethephon (ethylene-releasing agent) alone or in combination with 1-methylcyclopropene [1-MCP (ethylene perception inhibitor)] on leaf and mature fruit abscission of ‘Valencia’ sweet orange (Citrus sinensis) was studied. Leaf abscission increased and fruit detachment force (FDF) decreased significantly especially 4 days after ethephon treatment. Leaf drop rose to over 80% 7 days after application, whereas FDF was only 30% less than untreated control fruit. When 1-MCP was combined with ethephon and applied to ‘Valencia’ sweet orange canopies, leaf abscission was greatly reduced, but reduction in FDF proceeded unabated. We hypothesized that differential response of ‘Valencia’ sweet orange fruit and leaves to 1-MCP was correlated with expression of ethylene biosynthetic and signaling genes and their downstream action. Partial or full-length nucleotide sequences were obtained for ‘Valencia’ sweet orange homologs of 1-amino-cyclopropane-1-carboxylate synthase-1 (CsACS1), 2 (CsACS2), 1-amino-cyclopropane-1-carboxylate oxidase (CsACO), ethylene response sensor-1 (CsERS1), ethylene response-1 (CsETR1), 2 (CsETR2), 3 (CsETR3), constitutive triple response-1 (CsCTR1), ethylene insensitive-2 (CsEIN2), and ethylene insensitive 3-like-1 (CsEIL1) and 2 (CsEIL2). Ethephon application increased expression of biosynthesis genes CsACS1 and CsACO and receptors CsERS1 and CsETR2 in the abscission zones of leaves and mature fruit. Ethephon-induced increase in gene expression was completely suppressed by 1-MCP application in all but CsACS1 and CsACO in fruit abscission zones. Although gene expression was suppressed initially, CsACS1 and CsACO expression in fruit abscission zones treated with 1-MCP in the presence or absence of ethephon increased over the 7-day measurement period, suggesting that CsACS1 and CsACO expression were negatively regulated by basal ethylene production in this tissue. However, 1-MCP treatment alone did not loosen fruit, indicating that CsACS1 and CsACO played minor roles in fruit abscission. To determine if the difference in ethylene sensitivity was the basis of differential response to ethylene within the same organ, potted ‘Valencia’ sweet orange plants were treated with ethylene, and rates of blade and petiole drop and detachment forces at the laminar and petiolar abscission zones were studied. Although leaf blades abscised earlier than petioles, the force of detachment was similar, indicating no differences in ethylene sensitivity. Overall, the most significant difference between fruit and leaf abscission zones was seen in the expression of CsACS1 and CsACO genes, but the expression pattern was poorly correlated with abscission.

Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated

To investigate the regulatory mechanism(s) of ethylene biosynthesis in fruit, transgenic tomatoes with all known LeEIL genes suppressed were produced by RNA interference engineering. The transgenic tomato exhibited ethylene insensitivity phenotypes such as non-ripening and the lack of the triple response and petiole epinasty of seedlings even in the presence of exogenous ethylene. Transgenic fruit exhibited a low but consistent increase in ethylene production beyond 40 days after anthesis (DAA), with limited LeACS2 and LeACS4 expression. 1-Methylcyclopropene (1-MCP), a potent inhibitor of ethylene perception, failed to inhibit the limited increase in ethylene production and expression of the two 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) genes in the transgenic fruit. These results suggest that ripening-associated ethylene (system 2) in wild-type tomato fruit consists of two parts: a small part regulated by a developmental factor through the ethylene-independent expression of LeACS2 and LeACS4 and a large part regulated by an autocatalytic system due to the ethylene-dependent expression of the same genes. The results further suggest that basal ethylene (system 1) is less likely to be involved in the transition to system 2. Even if the effect of system 1 ethylene is eliminated, fruit can show a small increase in ethylene production due to unknown developmental factors. This increase would be enough for the stimulation of autocatalytic ethylene production, leading to fruit ripening.

Benzylaminopurine Application on Two Different Apple Cultivars (Malus domestica) Displays New and Unexpected Fruitlet Abscission Features

It has been previously shown that abscission of apple fruitlets is preceded by an increase in ethylene evolution and in the amount of transcripts for 1-aminocyclopropane-1-carboxylate oxidase (ACO), an enzyme catalysing the final step in ethylene biosynthesis. These events are concomitant with shedding induction and chemical thinning. There are several thinners but their mode of action and efficacy is poorly understood. One of them is benzylaminopurine (BA), a cytokinin believed to act by enhancing vegetative activity and stressing the competition between shoots and fruitlets, thus leading to fruitlet shedding. Nevertheless, the specific mechanism of action of BA and the variable effect depending on apple cultivar (easy or difficult to thin) are poorly understood. Abscission, the amount of MdACO1 transcripts and other parameters were followed in immature apple fruits during the period of physiological drop. The cultivars studied were 'Golden Delicious' and the 'spur' type 'Red Delicious'. BA was used as a thinning agent and was sprayed 14 d after petal fall (DAPF). Fruitlets were divided into central (C) and lateral (L) fruitlet populations. Fruitlet size was significantly different between C and L fruitlets but it did not differ much between the populations within the same cultivar. C fruitlets were characterized by basal ethylene evolution while L fruitlets displayed an increase in hormone biosynthesis during abscission induction. Cluster composition evaluated by the L/C ratio differed in the two varieties, being almost unchanged throughout abscission induction in 'Golden Delicious' and progressively decreasing in 'Red Delicious'. Shoot growth activity evaluated at the end of the season indicated a possible connection with both the ongoing abscission and BA application. MDACO1 transcripts were mainly detected in L fruitlets and the accumulation was related to total abscission in 'Golden Delicious', while in 'Red Delicious' expression was observed in both C and L fruitlets. BA probably exerts its thinning effect through vegetative growth. In the 'spur' type 'Red Delicious' the chemical is ineffective, probably due to a limited action on shoot growth due to genetic characteristics. The amount of MdACO1 transcripts in seeds is a good indicator of abscission.

Exogenous auxin effects on growth and phenotype of normal and hairy roots of Pueraria lobata (Willd.) Ohwi

Pueraria lobata hairy roots have faster elongationand more branches than normal roots. The responses of hairy roots and normalroots to treatment with three auxins, indole-3-acetic acid (IAA),indole-3-butyric acid (IBA), and naphthalene acetic acid (NAA) were different.In normal roots, all three auxins strongly stimulated lateral root formation atall tested concentrations. Responses to IAA and IBA in primary root growth andlateral root elongation were similar and depended on concentration; promotionat0.1 μM, no effect at 1.0 μM, and inhibition at2.5 μM. In hairy roots, lateral root formation varied inresponseto the different auxins, i.e. depressed by NAA, unaffected by IAA, and promotedby IBA. Primary root growth was slightly inhibited by IBA and was unaffected byIAA. However, mean lateral root length was reduced in response to IAA and IBA.Only NAA exerted strong inhibition on primary and lateral root elongation inboth root types. The similar free IAA and conjugated IAA content but quitedifferent basal ethylene production and biosynthesis in hairy and normal rootssuggested different mechanisms of response to exogenous auxins in the two roottypes.

Regulation of ethylene and polyamine synthesis by elevated carbon dioxide in cherimoya fruit stored at ripening and chilling temperatures

In this study we focused on the effect of high CO2 level (20%) on ethylene and polyamine biosynthesis in cherimoya (Annona cherimola Mill.) fruits stored at ripening (20˚C) and chilling (6˚C) temperatures. At ripening temperature, CO2 inhibited ethylene production, but 1-aminocyclopropane-1- carboxylate (ACC) oxidase activity was similar to that in ripe control fruits. CO2 treatment led to a decline in putrescine (Put) and a major accumulation of spermidine (Spd) and spermine (Spm) without any effect on arginine decarboxylase (ADC) activity. These results confirm the preferential transformation of Put to Spd and Spm in CO2 -treated fruits. At chilling temperature, the increase in ACC oxidase activity was inhibited and the Vmax of ADC increased. A combination of chilling temperature storage and high CO2 level led to suppression of basal ethylene production while ACC oxidase activity remained unchanged. In addition, fruits held at these conditions had higher polyamine titres than the untreated control. We propose that, in CO2 -treated fruits, the absence of autocatalytic or basal ethylene production, depending on the temperature, may be due to deviation of the S-adenosylmethionine (SAM) pool towards polyamine synthesis, primarily Spd and Spm.

Transport of 1-aminocyclopropane-1-carboxylic acid (ACC) in the transpiration stream of tomato (Lycopersicon esculentum) in relation to foliar ethylene production and petiole epinasty

We investigated the concentration and delivery of 1-aminocyclopropane-1-carboxylic acid (ACC) in the transpiration stream of flooded and well-drained 1-month-old tomato plants (Lycopersicon esculentum Mill. cv. Ailsa Craig) over time in parallel with foliar ethylene production and petiole epinasty. ACC was measured by gas chromatography using a nitrogen–phosphorus detector. Before analysis, roots of freshly detopped plants were pressurised pneumatically to make xylem sap flow at rates similar to those of whole plant transpiration. Delivery of ACC from roots to shoots of well-drained plants was sufficient to support basal ethylene production in shoots of unstressed plants. Delivery from flooded, oxygen-deficient, roots increased after 6 h and coincided with the onset of epinastic leaf curvature. Further increases in ACC delivery and epinastic curvature occurred later in the photoperiod. After 24 h flooding, ACC delivery in xylem sap was 28 times more than in well-drained plants. This increased export of ACC from flooded roots was more than sufficient to account for the extra ethylene production in the shoots and coincided with ACC accumulation in the leaves. Removing the shoot before flooding did not reduce ACC export from oxygen-deficient roots indicating that the ACC originated in roots and not the shoot. Increased ethylene production in petioles of flooded plants lagged 18 h behind epinasty.

Effects of floret maturity, cytokinin and ethylene on broccoli yellowing after harvest

Abstract The phytohormones ethylene and cytokinin have a central role in the postharvest yellowing of broccoli. The relationship between ethylene synthesis and the extent of yellowing was examined in three broccoli cultivars (‘Shogun’, ‘Green Belt’ and ‘Green Beauty’). In these cultivars, no consistent relationship was found between basal ethylene production and the rate of floret degreening (20 °C in darkness). In cultivar ‘Shogun’, floret maturity stages were determined by examining the developmental stages of the pollen. Different patterns of ethylene production, ethylene sensitivity, respiratory activity and chlorophyll loss occurred in florets of different maturities. Floret maturity is likely to have the greatest effect on the rate of broccoli yellowing. Dipping florets (cv. ‘Shogun’) in 6-benzylaminopurine (6-BA) stimulated ethylene production, depressed respiration rate and delayed floret yellowing. The effect of cytokinin on broccoli yellowing was related to cytokinin concentration and postharvest floret age when the cytokinin was applied. A continuous propylene treatment reduced the positive effect of cytokinin on broccoli colour changes.

Role of Ethylene in Axillary Shoot Proliferation of Lavandin—Interaction with Benzyladenine and Polyamines

The role of ethylene in in vitro axillary proliferation of lavandin (Lavandula officinalis Chaix x Lavandula latifolia Villars) was investigated. Basal ethylene production was modulated by the addition of exogenous growth regulators (BA and polyamines), ethylene precursor (ACC) and inhibitors (AVG and SA). The results indicate that BA action is mediated by ethylene and, furthermore, that ethylene can substitute for BA in shoot proliferation. Among tested polyamines (Put, Spd, Spn) only Put was effective in stimulating both the differentiation process and related ethylene production. Moreover, Put appears to have a regulatory function similar to BA. The data obtained in the present work outline the complexity of axillary budding which is under multihormonal control, ethylene playing an essential role.

Ethylene release from green spruce needles involves a combination of ACC-dependent and independent pathways

Aminoethoxyvinylglycine (AVG) and cobalt ions strongly inhibit the conversion of added methionine or aminocyclopropane-1-carboxylic acid (ACC) into ethylene by green-coloured, non-stressed Norway spruce (Picea abies L.) needles but only 30%–40% of basal ethylene formation is affected by such inhibitors. In addition, free radical-mediated ACC-independent ethylene formation (AIEF) of the type released by brown-coloured spruce needles also occurs in extracts from healthy green-coloured needles. Treatment with CdCl2 (10 mM), Na2S2O5 (5 mM) or FeSO4 (10 mM) induces 3–7 fold increases in the rates of ethylene evolution from green-coloured needles. However, only Cd2+-induced ethylene formation is inhibited by AVG while ethylene induced by S2O5 2- or Fe2+ is insensitive to added AVG although increased levels of ACC have also been detected in these treatments. Nevertheless, ethylene-forming decomposition of the precursors of AIEF is accelerated by S2O5 - or Fe2+ which indicates that the ethylene released from green-coloured spruce needles is formed by a combination of both the ACC-dependent and AIEF pathways.

Calcium regulation of exogenous and endogenous 1‐aminocylopropane‐1‐carboxylic acid bioconversion to ethylene

Ethylene production and overall levels of free and conjugated 1‐aminocyclopropane‐1‐carboxylic acid (ACC) were studied in parenchymatous tissues, excised from clmacteric apples (Malus domestica Borkh. cv. Granny Smith) and infiltrated with an incubation medium containing 0, 1, 10 or 100 mM Ca2+, with or without exogenous ACC (2 mM). Irrespective of whether exogenous ACC was applied or not, ethylene production was inhibited to the same extent (40%) by an apoplastic effect of 100 mM Ca2+. In the absence of external ACC, the inhibition was associated with an increase in total endogenous ACC and may be related to a reduction in the rate of the last step of ethylene pathway. This suggests that the ethylene‐forming enzyme (EFE) is localized in the plasma membrane. Low Ca2+ concentrations (1 mM) enhanced basal ethylene synthesis due to influx of Ca2+ into the cytosol, while overall concentrations of ACC in the tissue decreased. However, 1 mM Ca2+ did not stimulate ACC‐dependent ethylene formation. Thus, Ca2+ influx may stimulate the translocation of endogenous ACC from synthesis or storage compartment (s) to reactive site(s) of the plasma membrane. The concentration of 10 mM Ca2+ had no effect on basal ethylene production and appears to represent a balance point between the stimulating and inhibiting effects of 1 and 100 mM Ca2+, respectively, Furthermore, the charge‐times of exogenous ACC observed with 0, 1 and 10 mM Ca2+ suggest that EFE is located on the inner side of the plasma membrane.

The Chemotactic Peptide N-Formyl Methionyl-Loucyl-Phenylalanine Increases Mucosal Permeability in the Distal Ileum of the Rat

Activation of granulocytes within the lamina propria by luminally derived bacterial products may represent an important mechanism in the pathogenesis of inflammatory bowel disease. The objective of this study was to determine the effects of luminal perfusion with N-formyl methionyl-leucyl-phenylalanine (FMLP), a bacterial product that attracts and activates granulocytes, on mucosal permeability in different regions of the rat small intestine and colon. Mucosal permeability was measured using blood-to-lumen clearance of 51Cr-ethylenediaminetetraacetate during luminal perfusion with FMLP (10-3 to 10-8 M) dissolved in Tyrode's solution. Of the bowel segments studied, mucosal permeability was significantly increased by FMLP only in the distal 10 cm of ileum. The minimal FMLP concentration required to increase mucosal permeability was 10-6 M. The increased mucosal permeability induced by FMLP could be prevented by depletion of circulating granulocytes with antineutrophil serum. The greater sensitivity of the distal ileum to FMLP did not correlate with a higher tissue myeloperoxidase activity, but it was associated with a higher basal ethylene diaminetetraacetate clearance. These observations indicate that a high basal mucosal permeability to solutes the size of FMLP (5–6 Å radius), rather than a greater number of resident granulocytes in the lamina propria, predisposes the terminal ileum to the inflammatory actions of FMLP.

Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fruit development.

We have investigated the regulation of gene expression by the plant hormone ethylene by cloning mRNAs that accumulate in unripe tomato fruit (Lycopersicon esculentum) exposed to exogenous ethylene. The response to exogenous ethylene is rapid; within 30-120 min we detect an increase in the cloned mRNA concentrations. DNA sequence analysis indicates that one of the ethylene-inducible genes is related to a gene encoding wound-inducible proteinase inhibitor I. We have measured ethylene production during fruit development and detect low basal levels in unripe fruit and much higher levels in ripening fruit. Blot hybridization experiments show that expression of the cloned genes is developmentally regulated by ethylene during fruit ripening: the mRNAs produced by these genes are more abundant in ripe fruit than in unripe fruit, and this mRNA accumulation is repressed by a competitive inhibitor of ethylene action, norbornadiene. However, during fruit development some of the cloned mRNAs begin to accumulate when ethylene production is at a basal level, whereas other mRNAs begin to accumulate later when the endogenous ethylene concentration increases, suggesting that gene expression during fruit development can be activated by ethylene in two ways. In some cases gene expression is primarily activated by an increase in sensitivity to basal ethylene levels, whereas in other cases it may be regulated by an increase in ethylene concentration.

Ozone-induced ethylene release from leaf surfaces

Ozone-induced stress ethylene emissions from the adaxial and abaxial leaf surfaces of four plant species ( Glycine max [L.] Merr. cv. Dare, Lycopersicon esculentum Mill cv. Roma VF, Eucalyptus globulus Labill. and Hedera helix L.) were studied to determine if the stress ethylene diffused through the stomata or cuticle. In plants not exposed to ozone, basal ethylene was detected above both the adaxial and abaxial leaf surfaces of all the plant species examined, indicating that some ethylene can diffuse across the leaf cuticle. Ozone induced stress ethylene production in all species examined. Significant ozone-induced ethylene concentrations were detected above both surfaces of amphistomatous soybean ( Glycine) and tomato ( Lycopersicon) leaves. In contrast, ozone-induced ethylene production was associated only with the leaf surface (abaxial) that contained stomata for hypostomatous blue gum eucalyptus and English ivy ( Hedera) leaves; the leaf surface (adaxial) of the eucalyptus and ivy leaves which did not contain stomata did not release significant amounts of stress ethylene. These data indicate that ozone-induced stress ethylene primarily diffuses from the leaf via the stomata.

Production and Release of Ethylene from 1-aminocyclopropane-1-carboxylic Acid in Lemna minor L. in the Dark and at Different Carbon Dioxide Compensation Concentrations

Duckweed Lemna minor L. was used to investigate the in vivo conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in the dark and at different CO(2) compensation concentrations. The plants were incubated in closed bottles. Various constant CO(2) concentrations in the range 50 to 1000 ppm were obtained by applying different light intensities. During the incubation on ACC-containing solution (0.01 -1000 μM) in the dark, ACC accumulated in L. minor, and no saturation of ACC uptake occurred. ACC-dependent ethylene production, on the other hand, was saturated above 10 μM ACC (equivalent to 32 μM internal ACC). V(max) was 3.18 nmol h(-1) (g fresh wt)(-1) and apparent K(m) was 8.71 μM internal ACC. In the light, CO(2) below 1000 ppm was limiting for ACC-dependent ethylene production. In the presence of 1 and 10 μM ACC, half-maximum CO(2) activity was at 114 and 163 ppm, respectively. CO(2)-stimulation was very small at 0.1 μM external ACC and virtually absent in the case of basal ethylene production. Under non-limiting CO(2), ACC-dependent ethylene production rates in the light were higher than V(max) in darkness. The mechanism of COrstimulation of ACC-dependent ethylene production in the light was elucidated by comparing rates of ACC conjugation, and ethylene synthesis, release and retention under high and low CO(2). Inhibition of ethylene retention contributed the largest portion to the enhancement of ethylene emanation from L. minor under high CO(2) observed. However, a small stimulatory effect of CO(2) on ACC conversion to ethylene was also found, while the conjugation of ACC was not significantly affected by CO(2).