Effect of gibberellic acid on ACC content,EFE activity and ethylene release by floral parts of the senescing carnation flower

The application of gibberellic acid via the stem of intact preclimacteric carnation flowers inhibited the climacteric surge of ethylene evolution by the flowers. Gibberellic acid also inhibited the rate of ethylene production by all individual floral parts during both the early preclimacteric (low basal level of ethylene production) and the later climacteric stages of flower development. The extent of inhibition did however, vary from one floral part to another. The most pronounced inhibition was recorded in the petal bases between the preclimacteric and senescing stages. This suggests that the petal base is an important regulatory site for ethylene production and therefore may be involved in controlling the onset and degree of petal inrolling. In all floral parts endogenous levels of ACC were reduced with GA₃ treatment, being more pronounced in the petal bases. The potential of the flowers to convert applied ACC to ethylene was not deminished by gibberellic acid.Abbreviations GA₃ gibberellic acid - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

Synergistic effect of 1-aminocyclopropane-1-carboxylic acid and ethylene during senescence of isolated carnation petals

The effects of ethylene (C₂H₄), (2-chloroethyl)phosphonic acid (ethefon) and 1-aminocyclopropane-1-carboxylic acid (ACC) on senescence of isolated intact petals and of upper petal parts of carnation flowers ( Dianthus caryophyllus L. cv. White Sim) were investigated.
Isolated upper petal parts did not respond to treatment with ethefon or ACC. These tissues did, however, show severe wilting in intact petals that were treated with ethefon or ACC. When isolated upper petal parts were simultaneously treated with ACC and ethefon or ACC and ethylene, a marked synergistic effect on senescence was found. Treatment of isolated petals with radiolabeled ACC led to the accumulation of radiolabeled ACC and N-malonyl-ACC (MACC) in the upper parts. The formation of ethylene and the malonylation of ACC were inhibited by pretreatment of the flower with the inhibitor of ethylene action, silver thiosulphate (STS), which indicates that both were induced by endogenously produced ethylene. Treatment of isolated upper parts with ACC slightly increased their ethylene production. However, when these petal parts were simultaneously treated with ethylene and ACC, the conversion of ACC to ethylene was markedly stimulated.
The results indicate that, in intact petals, ethylene may be translocated from the basal to the upper part where it stimulates the activity of the ethylene-forming enzyme (EFE), thereby making the tissue receptive to ACC.
In addition, it was found that upon incubation of petal portions in radiolabeled ACC, both the petal tissue and the incubation solutions produced radiolabeled carbon dioxide. This was shown to be due to microorganisms that were able to metabolize the carbon atoms in the 2 and 3 position of ACC into carbon dioxide.     

Manipulation of ethylene biosynthesis

The plant hormone ethylene is involved in many plant processes ranging from seed germination to leaf and flower senescence and fruit ripening. Ethylene is synthesized from methionine, via S-adenosyl-L-methionine (SAM) and 1-amino-cyclopropane-1-carboxylic acid (ACC). The key ethylene biosynthetic enzymes are ACC synthase (ACS) and ACC oxidase (ACO). Manipulation of ethylene biosynthesis by chemicals and gene technology is discussed. Biotechnological modification of ethylene synthesis is a promising method to prevent spoilage of agricultural and horticultural products.     

Inhibition of ethylene biosynthesis in carnation petals by cytokinin

Pretreatment of detached carnation petals (Dianthus caryophyllus cv White Sim) for 24 hours with 0.1 millimolar of the cytokinins n⁶-benzyl-adenine (BA), kinetin, and zeatin blocked the conversion of externally supplied 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene and delayed petal senescence by 8 days. The normal enhanced wilting and increase in endogenous levels of ACC and ethylene production following exposure of petals to ethylene (16 μl/l for 10 hours), were not observed in BA-pretreated petals. In carnation foliage leaves pretreated with 0.1 mm BA, a reduction rather than inhibition of the conversion of exogenous ACC to ethylene was observed. This indicates that foliage leaves respond to cytokinins in a different way than petals. A constant 24-hour treatment with BA (0.1 mm) was not able to reduce ethylene production of senescing carnation petals, while 2 mm aminoxyacetic acid, a known inhibitor of ACC synthesis, or 10 mm propyl gallate, a free radical scavenger, decreased ethylene production significantly.     

Changes in protein and mRNA populations during the senescence of carnation petals

Developmental changes in polypeptide and mRNA popultions in carnation ( Dianthus caryophyllus L. cv. White Sim) petals were investigated during the senescence of harvested flowers. Total proteins were extracted from flower petals at various stages of senescence and subjected to separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis of the Coomassie blue stained gels revealed polypeptides with apparent molecular weights of 76, 62, 35.5 and 24 kDa which increased, while those with molecular weights of 70.5, 67.5, 46.5 and 31 kDa decreased during petal senescence. Changes in mRNA populations were investigated by translating poly (A)⁺RNA, isolated from carnation petals, in vitro using the rabbit reticulocyte lysate system. Polypeptides synthesized in vitro were separated by one- and two-dimensional gel electrophoresis and visualized by fluorography. Three classes of mRNA's were associated with the senescence of carnation petals. The majority of the mRNA's were constitutive at all stages of senescence. Another class of mRNA's increased with the climacteric rise in ethylene production, which accompanied the onset of senescence. Their translation products were 81, 58, 42, 38 and 35 kDa. In addition, several mRNA's appeared to decrease in abundance during the course of petal senescence. These results indicate that senescence of carnation flower petals is associated with changes in gene expression.     

Flotation of lipid-protein particles containing triacylglycerol and phospholipid from the cytosol of carnation petals

Lipid-protein particles ranging from 20 to 250 nm in diameter have been isolated from the cytosol of carnation petals by flotation centrifugation and also by ultrafiltration. The cytosolic lipid-protein particles resemble oil bodies, lipid-protein particles found in oil-bearing seeds, in that they contain triacylglycerol, are circumscribed by phospholipid that is not organized in a bilayer, appear to be derived from membranes and can be isolated by flotation. However, the cytosolic particles are distinguishable from oil bodies in that triacylglycerol is not the dominant lipid. Indeed, they contain a spectrum of lipids in addition to phospholipids and triacylglycerol including free fatty acids, sterol and wax esters, phosphatidic acid and diacylglycerol. These same lipids are present in corresponding microsomal membranes as well, but in much smaller proportions relative to phospholipid. The lipid-protein particles from carnation petals contain a 17-kDa protein that is of similar size to oil body oleosin, but does not cross-react with anti-oleosin antibodies. The data indicate that these cytosolic particles are structurally and chemically similar to oil bodies and are consistent with the notion that their genesis may be a means of removing destabilizing lipids from membrane bilayers.     

Recovery of ethylene biosynthesis in diazocyclopentadiene (DACP)-treated tomato fruit

Diazocyclopentadiene (DACP), a competitive ethylene action inhibitor binds irreversibly to the ethylene receptor to reduce tissue responses to ethylene. Tomato fruit (Lycopersicon esculentum Mill cv lsquo;Rondellorsquo;) were treated with DACP at the mature green stage. Ethylene biosynthesis and respiration rate were depressed. Color changes from green to red were delayed. Compared to the control, ACC content increased and ACC oxidase activity in vivo decreased in DACP-treated fruit. Thus, decrease of ethylene production caused by DACP treatment was due to the reduction of ACC oxidase activity. The decline in ripening subsequently recovered after DACP treatment. Results from the Northern analysis for gene expression of ACC synthase and ACC oxidase, showed that expression of both genes declined in DACP-treated fruit, and then recovered. Therefore the recovery of ethylene production was due to the recovery in gene expression and activity of ACC oxidase. We conclude that the effects of DACP on ethylene biosynthesis are on expression of ACC synthase and ACC oxidase genes, and/or regulation of ACC oxidase activity.     

The site of 1-aminocyclopropane-1-carboxylic acid synthesis in senescing carnation petals

To study the cause of the uneven production of ethylene by upper and basal portions of detached petals of carnation ( Dianthus caryophyllus L. cv. White Sim), the petals were divided and exposed to ethylene (30 μl 1^-1 for 16 h). The treatment induced rapid wilting and autocatalytic ethylene production in the basal portion similar to that induced in entire petals. In contrast to the response in entire petals and the basal portions, the upper portions responded to ethylene by delayed wilting and much lower ethylene production. Aminocyclopropane carboxylic acid (ACC)-synthase activity in the basal portion of the petals was 38 to 400 times that in the upper portion. In untreated detached petal pieces from senescing carnation flowers, ethylene production by the upper portion declined after 6 h while the basal portion was still producing ethylene at a steady rate 18 h later. Application of ACC to the upper portion of senescing petals increased their ethylene production. α-Aminooxyacetic acid (0.5 m M ), reduced the ethylene production of the senescing basal portion more than that of the upper portion. Endogenous ACC content in basal portions of senescing carnation petals was 3 to 4 times higher than in the upper parts. When detached senescing petals were divided immediately after detaching, the endogenous ACC levels in upper portions remained steady or declined during 24 h after division, while in the basal portions the ACC level rose steadily as in the intact petals. There was no change in the conjugated ACC in either portion after 24 h. Benzyladenine (BA) applied as a pretreatment to entire preclimacteric petals greatly reduced the development of ACC-synthase activity of the basal portion, but had little effect on the activity in the upper portion of the petal. In both portions, however, BA effectively reduced the conversion of ACC to ethylene.     

The involvement of ethylene in liatris corm dormancy

Corms of liatris (L. spicata, cv. Callilepsis) show a seasonal dormancy, being most active in the November harvest and least active in June. Storage of dormant corms at 3 °C for about 9 weeks resulted in a complete break of dormancy. This was accompanied by a sharp temporal increase in their rate of ethylene production, which was more pronounced in the buds than in the parenchyma tissue. Application of ethylene to the corms in the form of ethrel solution increased both ethylene production rate and sprouting. The ethylene-forming activity from ACC, measured both in vivo and in vitro, was higher in corms producing more ethylene. However, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) of the corms was inversely related to their ethylene production rate. Ethylene thus seems to be involved in the dormancy control of liatris corms, and its production is apparently regulated mainly by the activity of the membranous ethylene-forming system.     

Effect of gibberellic acid on carnation flower senescence: evidence that the delay of carnation flower senescence by gibberellic acid depends on the stage of flower development

Gibberellic acid at concentrations of 10^–5 M and 10^–4 M delayed the senescence of cut carnation flowers, when applied continuously via the stem, to flowers between the closed brush and fully open stages of development. Older flowers with reflexed petals were unresponsive. Treatment with paclobutrazol, an inhibitor of GA biosynthesis, prevented tight buds from opening fully, reduced the longevity of partially open flowers, but was ineffective when applied continuously to fully open flowers. Gibberellic acid-treated flowers did not show simultaneous petal inrolling, a known indicator of senescence, and the time to complete petal drying was extended. Gibberellic acid modified the climacteric ethylene rise in a manner consistent with the extension of longevity. These results provide evidence for a correlative role of gibberellins in flower development.Abbreviations GA₃ gibberellin A₃ - GLC gas liquid chromatography     

Effect of short-chain fatty acids on the ethylene pathway in embryonic axes of Cicer arietinum during germination

The effect of short-chain saturated fatty acids (C₅–C₁₀) on the biosynthesis of ethylene in embryonic axes of chick-pea ( Cicer arietinum L.) seeds was investigated. The emergence of radicle and fresh weight of embryonic axes diminished with increasing number of carbons. The inhibition of germination caused by lower concentrations (1 m M ) of fatty acids (C₅–C₁₀) was partially reversed by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), whereas exogenous ethylene was able to overcome the inhibitory effect provoked by all concentrations (1–5 m M ) of applied fatty acids (C₅–C₁₀). Ethylene production rates, and enzyme activities of ACC synthase and ACC oxidase decreased concomitantly with the molecular mass and increasing concentration of fatty acids. The inhibitory effect of these acids on ethylene production seems to result not only from a decreased ACC synthesis, but also from an enhancement of 1-malonylamino)cyclopropane-1-carboxylic acid (MACC) synthesis.     

Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought‐stress conditions

A transgenic gene‐silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down‐regulated ACC synthases (ACSs), enzymes that catalyse the rate‐limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought‐stress and rain‐fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype × transgene) × environment interaction existed among the events, highlighting the need to better understand the context in which the down‐regulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis‐silking interval and a concomitant increase in kernel number/ear in transgene‐positive events versus nulls. Selected events were also field tested under a low‐nitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/acre) yield increase. This set of extensive field evaluations demonstrated that down‐regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.     

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

Chilling-induced ethylene biosynthesis in Braeburn apples

We observed a chilling-induced ethylene biosynthesis in Braeburn apples.The stimulatory effect depended on the length of the cooling period. The longerthe period, the stronger the stimulation. Low temperature stimulated activityand gene expression of ACS, but only stimulated gene expression of ACO. Thestimulatory effect of low temperature on gene expression was stronger andearlier in ACS than in ACO. 1-MCP (1-methylcyclopropene), an inhibitor ofethylene action, inhibited ethylene biosynthesis in fruit stored at 20°C and 0 °C. This inhibitory effect can beslightly recovered in fruit stored at 0 °C, but not at 20°C. Expression of genes for ACS and ACO was weaker in1-MCP-treated fruit stored at 20 °C, than those at 0°C. Thus, it is possible that expression of genes for ACS andACO in fruit at low temperature was mainly, but not completely, regulated bytheethylene receptor.     

De-submergence-induced ethylene production in Rumex palustris: regulation and ecophysiological significance

Rumex palustris responds to total submergence by increasing the elongation rate of young petioles. This favours survival by shortening the duration of submergence. Underwater elongation is stimulated by ethylene entrapped within the plant by surrounding water. However, abnormally fast extension rates were found to be maintained even when leaf tips emerged above the floodwater. This fast post-submergence growth was linked to a promotion of ethylene production that is presumed to compensate for losses brought about by ventilation. Three sources of ACC contributed to post-submergence ethylene production in R. palustris: (i) ACC that had accumulated in the roots during submergence and was transported in xylem sap to the shoot when stomata re-opened and transpiration resumed, (ii) ACC that had accumulated in the shoot during the preceding period of submergence and (iii) ACC produced de novo in the shoot following de-submergence. This new production of ethylene was associated with increased expression of an ACC synthase gene (RP-ACS1) and an ACC oxidase gene (RP-ACO1), increased ACC synthase activity and a doubling of ACC oxidase activity, measured in vitro. Out of seven species of Rumex examined, a de-submergence upsurge in ethylene production was seen only in shoots of those that had the ability to elongate fast when submerged.     

Invertase inhibitor-control of sucrose transport from carnation petals to other flower parts

An investigation was made of the distribution of metabolites in the receptacles and ovaries, and the activity of an invertase inhibitor after feeding the petals with ¹⁴C-sucrose and cycloheximide during the natural aging of cut carnation flower. The cycloheximide treatment blocked the synthesis of the invertase inhibitor in petals and maintained a relatively high level of invertase activity during flower senscence. Treatment with cycloheximide decreased the movement of radioactivity from petals to receptacles and ovaries from 7 to 10 times, compared to non-treated control flowers. IAA applied on the stigma had no influence on synthesis of the inhibitor and distribution of the radioactivity.     

Ethylene biosynthesis in tissues of young and mature avocado fruits

Sitrit, Y., Blumenfeld, A. and Riov, J. 1987. Ethylene biosynthesis in tissues of young and mature avocado fruits.
Avocado (Persea americana Mill.) fruit tissues differ greatly in their capability to pro duce wound ethylene. In fruitlets, the endosperm lacks the ability to produce ethylene because no 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized and no activity of the ethylene-forming enzyme (EFE) is present. The cotyledons (embryo) do not produce significant amounts of ethylene at any of the developmental stages of the fruits, although in both young and mature fruits they contain a relatively high level of ACC synthase (EC 4.4.1.-) activity. Because of the very low EFE activity present in the cotyledons, most of the ACC formed in this tissue is conjugated. Of the various fruitlet tissues, the seed coat has the highest potential to produce ethylene. This is due to a high ACC synthase activity and particularly a high EFE activity. Also, the seed coat is very sensitive to the autocatalytic effect of ethylene. Fruitletpericarp possesses a lower potential to produce ethylene than the seed coat. Towardruit maturiy, the endosperm disappears and the seed coat shrivels and dies so that the pericarp and the cotyledons remain as the only active tissues in the mature fruit. At this stage, the pericarp is the only tissue producing ethylene. Mature precli macteric pericarp has a lower potential to produce ethylene than fruitlet pericarpThe role of ethylene in regulating various physiological processes at different stages of fruit maturation is discussed.