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.     

Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene

The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1-aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1-carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric.     

Sites of ethylene production in the pollinated and unpollinated senescing carnation (Dianthus caryophyllus) inflorescence

Production of endogenous ethylene from the styles, ovary and petals of pollinated and unpollinated flowers of Dianthus caryophyllus L. was measured. The rate of ethylene production of cut, unpollinated flowers aged in water at 18°C was low until the onset of petal wilting, when a rapid surge of ethylene occurred in all tissues. The flower ethylene production was evolved mostly from the styles and petals. The bases of petals from unpollinated, senescing flowers evolved ethylene faster and sometimes earlier than the upper parts. Treatment of cut flowers with propylene, an ethylene analogue, accelerated wilting of flower petals and promoted endogenous ethylene production in all flower tissues. Pollination of intact flowers also promoted endogenous ethylene production and caused accelerated petal wilting within 2–3 days from pollination. Although the data are consistent with the hypothesis that ethylene forms a link between pollination of the style and petal wilting, in the unpollinated flower the style and petals can evolve a surge of ethylene independently of each other, about the time when the petals irreversibly wilt. The results are discussed in relation to the role of ethylene in flower senescence.     

Role of short-chain saturated fatty acids in the control of ethylene sensitivity in senescing carnation flowers

In cut carnations ( Dianthus caryophyllus L. cv. Cally). petal senescence was associated with a climacteric pattern in ethylene production and an increase in ethylene sensitivity during the preclimacteric stage. The increase in ethylene sensitivity was caused by short-chain saturated fatty acids (C₇ to C₁₀) produced in the petals during the early stages of senescence. Pollination or application of octanoic acid to the styles of unpollinated flowers resulted in a sudden increase in ethylene sensitivity and a marked acceleration of senescence. Treatment with silver thiosulfate (STS) resulted in a suppression of ethylene sensitivity and a marked reduction in the levels of these fatty acids. However, even in STS-treated flowers pollination or treatment with octanoic acid gave rise to a drastic increase in ethylene sensitivity. Exposure of carnation flowers to 2. 5-norbornadicne (NBD) vapours resulted in a dramatic suppression of ethylene sensitivity which was also overridden by stylar application of octanoic acid. Exposure to NBD suppressed the increase in ethylene sensitivity caused by treatment with octanoic acid. It appears that short-chain saturated fatty acids increased ethylene sensitivity by increasing the ability of the tissue to bind ethylene.     

Regulation of ethylene biosynthesis in response to pollination in tomato flowers

Pollination of many flowers leads to an increase in ethylene synthesis and flower senescence. We have investigated the regulation of pollination-induced ethylene synthesis in tomato (Lycopersicon esculentum) using flowers of the dialytic (dl) mutant, in which pollination can be manipulated experimentally, with the aim of developing a model system to study tomato flower senescence. Ethylene synthesis increased rapidly in dl pistils following pollination, leading to accelerated petal senescence, and was delayed in ethylene-insensitive Never-ripe (Nr) pistils. However, Nr pistils eventually produced more ethylene than dl pistils, suggesting the presence of negative feedback regulation of ethylene synthesis following pollination. LEACS1A expression correlated well with increased ethylene production in pollinated dl pistils, and expression in Nr revealed that regulation is via an ethylene-independent mechanism. In contrast, the induction of the 1-aminocyclopropane-1-carboxylic acid oxidases, LEACO1 and LEACO3, following pollination is ethylene dependent. In addition, the expression profiles of ACS and ACO genes were determined during petal senescence and a hypothesis proposed that translocated 1-aminocyclopropane-1-carboxylic acid from the pistil may be important for regulating the initial burst of ethylene production during petal senescence. These results are discussed and differences between tomato and the ornamental species previously studied are highlighted.     

Senescence in isolated carnation petals : effects of indoleacetic Acid and inhibitors of protein synthesis

Indoleacetic acid induces senescence in isolated carnation (Dianthus caryophyllus, cv. White Sim) petals, increasing the duration and amount of ethylene production. This effect is inhibited by Actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, a translational inhibitor of protein synthesis. The ability of petals to respond to indoleacetic acid appears to be a function of physiological age. Indoleacetic acid is capable of enhancing ethylene evolution and senescence only in specific portions of the petal.     

Senescence in cut carnation flowers: Temporal and physiological relationships among water status, ethylene, abscisic acid and membrane permeability

Changes in water status, membrane permeability, ethylene production and levels of abscisic acid (ABA) were measured during senescence of cut carnation flowers ( Dianthus caryophyllus L. cv. White Sim) in order to clarify the temporal sequence of physiological events during this post-harvest period. Ethylene production and ABA content of the petal tissue rose essentially in parallel during natural senescence and after treatment of young flowers with exogenous ethylene, indicating that their syntheses are not widely separated in time. However, solute leakage, reflecting membrane deterioration, was apparent well before the natural rise in ethylene and ABA had begun. In addition, there were marked changes in water status of the tissue, including losses in water potential (ψ_w), and turgor (ψ_p), that preceded the rise in ABA and ethylene. As senescence progressed, ψ_w continued to decline, but ψ_p returned to normal levels. These temporal relationships were less well resolved when senescence of young flowers was induced by treatment with ethylene, presumably because the time-scale had been shortened. Thus changes in membrane permeability and an associated water stress in petal tissue appear to be earlier symptoms of flower senescence than the rises in ABA or ethylene. These observations support the contention that the climacteric-like rise in ethylene production is not the initial or primary event of senescence and that the rise in ABA titre may simply be a response to changes in water status.     

香石竹花瓣对乙烯的敏感性与蛋白质合成

基因转录抑制剂α-amanitin和蛋白质合成抑制剂cycloheximide完全抑制了香石竹(Dianthuscaryophyllus L.cvs.White Sim and Sandrosa)花瓣对乙烯反应的症状,包括花瓣卷曲和细胞膜离子渗漏增加。观察到花中蛋白质合成能力随着花的衰老而降低,花对乙烯的敏感性随花的衰老而增加。但是用乙烯合成抑制剂aminooxyacetic acid(AOA)预处理切花,则改变了花对乙烯敏感性的变化趋势。常用的香石竹品种D.caryophyllus L.cv.White Sim花经AOA处理后,对乙烯的敏感性随着花的衰老而下降。这些结果揭示花对乙烯的敏感性可能受蛋白质合成能力影响。     

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.     

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.     

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.     

Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

The Involvement of Water Stress and Ethylene in Senescence of Cut Carnation Flowers

Exposing cut carnation (Dianthus caryophyllus, cv. White Sim)to short term (12 h) water stress resulted in a marked increasein the water saturation deficit (WSD) of the petals. Full recoveryoccurred upon transfer of the flowers to water in humid conditions(r.h. 85%). However, an increase in aminocyclopropane carboxylicacid (ACC) content occurred immediately upon stress. An associatedrise in ethylene production following transfer to humid conditionswas observed earlier than in the control. Exogenous ethylene,applied alone or in combination with water stress, increasedthe WSD of the petals. Continuous treatment of cut flowers with amino-oxyacetic acid(AOA), a known inhibitor of ACC synthesis, suppressed ethyleneproduction, delayed the rise in WSD which accompanied developmentand senescence and hence delayed wilting. Similar results wereobtained with short term (2 h) treatment with AOA prior to stressingthe flowers. Short term AOA treatment partially inhibited therise in WSD during the stress period. On the basis of our findings, in particular that no rise inethylene production occurred during water stress, it is suggestedthat the effect of water stress is not directly mediated byethylene. The possible modulatory effect of water stress andAOA on certain characteristics of the petal cell membrane isdiscussed.     

Comparison between Ultraviolet Irradiation and Ethylene Effects on Senescence Parameters in Carnation Flowers

The effects of ethylene and ultraviolet (UV) irradiation on parameters of senescence in carnation (Dianthus caryophyllus L. cv White Sim) flowers were characterized and compared.

UV irradiation (λ_max = 254 nanometers), at fluences above 18 kilojoules per square meter, induced petal in-rolling, similar to that which occurred during natural senescence or after ethylene treatment. Increase in the UV dose from 36 to 54 kilojoules per square meter increased the rate of in-rolling to a maximum. Petal in-rolling was accompanied by increased electrolyte leakage, whether it occurred during natural senescence or was induced by UV irradiation or ethylene.

Sucrose uptake by cells, membrane ATPase activity, and membrane lipid fluidity all decreased after UV treatment. These parameters were shown earlier to decline during natural or ethylene-induced senescence.

UV irradiation induced ethylene production by the petals only during the period of irradiation. However, silver thiosulfate treatment, which blocks ethylene action, showed that the irradiation effects were not due to the ethylene evolved.

On the basis of the above results, we concluded that both ethylene and UV irradiation promote a sequence of reactions in the tissue similar to those of natural senescence. However, UV irradiation initiates a reaction which is independent of that which ethylene initiates.

     

Roles of pollination and short-chain saturated fatty acids in flower senescence

Pollination induced an immediate increase in ethylene production in Dianthus caryophyllus and Petunia hybrida. In Cymbidium, a lag of several hours was observed. In all three species, pollination induced premature flower senescence. Treatment of the stigmatic surface with aminoethoxyvinylglycine prior to pollination effectively blocked the increase in ethylene production and alleviated the detrimental effect of pollination on flower life.In all three tested species, octanoic and decanoic acids, when applied to the stigmatic surface, had no effect on ethylene production and flower life. In isolated Cymbidium lips placed with their cut base in solutions containing these fatty acids, no effects on red colouration, ethylene production, and ethylene forming enzyme activity were observed. In addition, ethylene sensitivity of isolated lips was not affected. The putative regulatory role of short-chain saturated fatty acids in (pollination-induced) flower senescence is discussed.     

Unusual ethylene-related behavior in senescing flowers of the carnation Sandrosa

The time course of ethylene production by senescing carnation ( Dianthus caryophyllus L. cv. Sandrosa) flowers was studied. These flowers are unusual in that they do not exhibit an autocatalytic increase in ethylene production nor do they develop petal in-rolling. Exposure of the flowers to exogenous ethylene resulted in a rise in their ethylene-forming enzyme (EFE) activity and ethylene production, and at the same time a marked decline in their fresh weight. Natural senescence was also accompanied by a rise in EFE activity, but with no concomitant rise in 1-amino cyclopropane carboxylic acid synthase activity nor in ethylene production. A shift in responsiveness to ethylene was observed, with young flowers more responsive to exogenous ethylene than older flowers. The results are discussed in terms of a proposed mechanism allowing for the decline in competence of this cultivar to respond to ethylene during senescence.