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.     

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity.Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.     

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.     

The Relationship Between the Change of Calmodulin Content and Ethylene Biosynthesis of Carnation Flower During Its Senescence

The relationship between the change of calmodulin content and the ethylene hiosynthesis in cut carnation flower (Dianthus caryophyllus ‘sun besm’ ) during its senescence was studied. Ethylene releasing was detected at the forth day and reached its peak at the sixth day after the cut carnation flower was cultured under controlled conditions of 27 ℃ with a 14 h photoperiod of 15000 lx provided by fluorescent lamps. The change of calmodulin content positively correlated with the increased' content of ACC, the activity of ACC synthase and ethylene production. The calmodulin contents in petals of the detached flower treated with GA, silver thiosulfate (STS) and aminooxyacetic acid (AOA) were lower than those in control flower petals before they withered, ethylene releasing was reduced and their senescence was delayed too. Ca2+ stimulated ethylene releasing in carnation flower petals, but chlorobenzene (CPZ), an antagonist of calmodulin, inhibited its releasing. It seemed that calmodulin was involved in the regulation of senescence of carnation flower.     

Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.)

Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

Ethylene and flower senescence

The end of the relatively short life of carnations held in air is associated with climacteric rises in ethylene production and respiration, and coordinate rises in activity of the enzymes of the ethylene biosynthetic pathway. Carnation sensescence is associated with derepression of specific genes, increased polyribosome activity, and major changes in patterns of protein synthesis. Isotopic competition assays indicate the presence in carnation petals of ethylene binding activity with the expected characteristics of the physiological ethylene receptor. Inhibition of ethylene production and/or ethylene binding (whether in selected varieties, or by treatment with chemicals) results in longer-lived carnations. Examination of other flowers shows that the carnation is not a universal paradigm for flower senescence. The response to ethylene varies widely, and in many species petal wilting occurs without any apparent involvement of ethylene.     

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 μl l⁻¹. Levels up to 1000 μl l⁻¹ do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 μl l⁻¹. At least 1 μl l⁻¹ ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO₂ absorbant, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.     

1-aminocyclopropane-l-carboxylic acid (ACC)—The transmitted stimulus in pollinated flowers?

Ethylene production and senescence of petals of pollinated carnation flowers were not prevented by removal of the ethylene produced by the gynoecium, suggesting that these events are a response to movement from the gynoecium of some stimulus other than ethylene gas. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to the stigmas caused an initial increase in gynoecium and petal ethylene production similar to that reported for pollinated flowers. This response was not seen in flowers whose stigmas were treated with indoleacetic acid (IAA). When [2-¹⁴C]ACC was applied to the stigmas of carnation flowers, radioactive ethylene was produced both by the gynoecia and by the petals. The possibility that ACC, transported from the stigmas to the petals, is responsible for the postpollination changes in carnation flowers is discussed.     

Conditioning period, CO2 and GR24 influence ethylene biosynthesis and germination of Striga hermonthica

Germination of witchweed ( Striga hermonthica [Del.] Benth), an important root parasite on poaceous crops, requires pretreatment 'conditioning' in a warm moist environment and a subsequent exposure to a stimulant. The roles of conditioning period, CO₂ and a strigol analogue (GR24) in ethylene biosynthesis and germination of the parasite were investigated. Conditioning increased the seeds' capacity to oxidize exogenous 1-aminocyclopropane-1-carboxylic acid (ACC). Exogenous CO₂ increased the seeds capacity to oxidize ACC by 3- to 9-fold. A combination of GR24 and ACC increased ethylene production by more than 3-fold in comparison with the rates obtained using these compounds separately. Aminoethoxyvinylglycine (AVG) completely inhibited ethylene induction by GR24, but not by ACC. A GR24 treatment, made subsequent to conditioning in GR24, did not induce ethylene. However, seeds conditioned in GR24 and then given 1 m M ACC produced 293 nl l⁻¹ ethylene. ACC oxidase (ACCO) activity in crude extracts was increased by conditioning and CO₂. The enzyme displayed an absolute requirement for ascorbate. Absence of exogenous Fe²⁺ reduced enzyme activity only by 14%. GR24 applied during conditioning reduced germination in response to a subsequent GR24 treatment. ACC was, invariably, less effective in inducing S. hermonthica germination than GR24 even at concentrations which induce more ethylene than concurrent GR24 treatments. The results are consistent with a model in which conditioning removes a restriction on the ethylene biosynthetic pathway in S. hermonthica seeds. GR24 modulates the key enzymes in ethylene biosynthesis. The stimulant suppresses ethylene biosynthesis in unconditioned seeds and promotes it in conditioned ones. Germination of S. hermonthica results from the joint action of GR24 and the ethylene it induces.     

Role of ethylene in the senescence of isolated hibiscus petals

Senescence of petals isolated from flowers of Hibiscus rosa-sinensis L. (cv Pink Versicolor) was associated with increased ethylene production. Exposure to ethylene (10 microliters per liter) accelerated the onset of senescence, as indicated by petal in-rolling, and stimulated ethylene production. Senescence was also hastened by basal application of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminooxyacetic acid, an inhibitor of ethylene biosynthesis, effectively inhibited ethylene production by petals and delayed petal in-rolling. In marked contrast to these results with mature petals, immature petals isolated from flowers the day before flower opening did not respond to ethylene in terms of an increase in ethylene production or petal in-rolling. Furthermore, treatment with silver thiosulfate the day before flower opening effectively prevented petal senescence, while silver thiosulfate treatment on the morning of flower opening was ineffective. Application of ACC to both immature and mature petals greatly stimulated ethylene production indicating the presence of an active ethylene-forming enzyme in both tissues. Immature petals contained less free ACC than mature, presenescent petals and appeared to possess a more active system for converting ACC into its conjugated form. Thus, while the nature of the lack of responsiveness of immature petals to ethylene is unknown, ethylene production in hibiscus petals appears to be regulated by the control over ACC availability.     

Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers

Pollination of flowers of standard carnation (Dianthus caryophyllus L. cv. White Sim) with pollen from flowers of miniature carnations (D. caryophyllus L. cv. Exquisite) caused them to wilt irreversibly within 1 to 2 days. Pollination stimulated a sequential increase in ethylene production by stigmas, ovaries, receptacles, and petals of the flowers. The ACC content of the stigmas increased rapidly in the first few hours after pollination. The possibility that subsequent production of ethylene by other parts of the flower is stimulated by translocated ACC is discussed. Ethylene production and ACC content of other parts of the flower reached their maximum 24 h after pollination. The petal tissues contributed the bulk of the ethylene productionper flower thereafter. There appears to be a qualitative difference between the enzyme in the stigmas converting ACC to ethylene and that in other parts of the flower.     

In vitro growth and ripening of strawberry fruit in the presence of ACC, STS or propylene

Strawberry ( Fragaria ananassa Duch.) fruit exhibit limited capacity for continued development following harvest. This problem can be circumvented by maintaining harvested strawberry fruit in solutions containing sucrose and a bactericide. In this study, we investigated the respiratory and ethylene production kinetics and ethylene responsiveness in strawberry fruit harvested immature and ripened in vitro in the presence of propylene. The effects of 1-amino-cyclopropane-1-carboxylic acid (ACC) and silver thiosulfate (STS) alone and in combination were also examined. Respiration and ethylene patterns of fruit harvested green and developed in vitro declined with maturation and ripening, as did those of field-grown fruit harvested at different stages of ripeness. Exposure of detached green strawberry fruit to 5000 μl litre^-1 propylene failed to stimulate respiration or ethylene production, but advanced pigmentation changes and fresh-weight gain significantly. Excised fruit provided with 1 mol^-3 ACC exhibited increased ethylene production, enhanced fresh-weight gain, and accelerated anthocyanin accumulation, but showed no change in respiration. The developmental response of harvested strawberry fruit to propylene or ACC was dependent on fruit maturity at harvest, with white fruit exhibiting greater insensitivity compared with green fruit. Silver thiosulfate (0.5 mol^-3) applied alone or in combination with ACC failed to delay ripening in excised strawberry fruit. These experiments demonstrate that ripening in detached strawberry fruit can be modified by ethylene only in green fruit that are provided with a carbohydrate source. Ethylene, when applied exogenously as ACC or propylene to green fruit, can slightly increase fruit growth and the rate of colour development.