The role of ethylene in the senescence of carnation flowers, a review

The senescence of flower petals is a highly regulated developmental process which requires active gene expression and protein synthesis. The biochemical changes associated with petal senescence in carnation flowers include an increase in hydrolytic enzymes, degradation of macro-molecules, increased respiratory activity and a climacteric-like increase in ethylene production. It is clear that the gaseous phytohormone ethylene plays a critical role in the regulation and coordination of senescence processes. Many reviews on physiology and mode of action of ethylene are available. Molecular cloning led to the isolation of genes involved in ethylene biosynthesis and action. This review describes the current status of the studies on regulation of ethylene biosynthesis and ethylene response in carnation flowers. An overview is given of studies on senescence-related gene expression and possibilities to improve postharvest longevity by genetic engineering.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -amino-isobutyric acid - AOA amino oxyacetic acid - AVG aminoathoxyvinyl glycine - DACP diazocyclopentadiene - EFE ethylene forming enzyme - MACC malonyl 1-aminocyclopropane-1-carboxylic acid - MTA 5-methylthio-adenosine - NBD 2,5 norbornadiene - ppb parts per billion - SAM S-adenosyl-methionine - STS silver thiosulphate     

Effect of 2,5-Norbornadiene upon Ethylene Biosynthesis in Midclimacteric Carnation Flowers

The climacteric increase in ethylene production in carnation (Dianthus caryophyllus L. cv White Sim) flowers is known to be accompanied by an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase and ethylene forming enzyme (EFE) activities. When midclimacteric flowers were exposed to 2,5-norbornadiene, which blocks ethylene action, ethylene production began to decrease after 2 to 3 hours. ACC synthase activity was markedly reduced after 4 hours and the increase in EFE activity was blocked indicating that the autocatalytic signal associated with ethylene action stimulates both enzyme activities.     

Role of the gynoecium in natural senescence of carnation (Dianthus caryophyllus L.) flowers

Although the role of the gynoecium in natural senescence of the carnation flower has long been suggested, it has remained a matter of dispute because petal senescence in the cut carnation flower was not delayed by the removal of gynoecium. In this study, the gynoecium was snapped off by hand, in contrast to previous investigations where removal was achieved by forceps or scissors. The removal of the gynoecium by hand prevented the onset of ethylene production and prolonged the vase life of the flower, demonstrating a decisive role of the gynoecium in controlling natural senescence of the carnation flower. Abscisic acid (ABA) and indole-3-acetic acid (IAA), which induced ethylene production and accelerated petal senescence in carnation flowers, did not stimulate ethylene production in the flowers with gynoecia removed (-Gyn flowers). Application of 1-aminocyclopropane-1-carboxylate (ACC), the ethylene precursor, induced substantial ethylene production and petal wilting in the flowers with gynoecia left intact, but was less effective at stimulating ethylene production in the -Gyn flowers and negligible petal in-rolling was observed. Exogenous ethylene induced autocatalytic production of the gas and petal wilting in the -Gyn flowers. These results indicated that ethylene generated in the gynoecium triggers the onset of ethylene production in the petals of carnation during natural 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.     

Inhibition of wilting and autocatalytic ethylene production in cut carnation flowers by cis-propenylphosphonic acid

The effect of cis-propenylphosphonic acid (PPOH), a structural analoge of ethylene, on flower wilting and ethylene production was investigated using cut carnation flowers which are very sensitive to ethylene. Wilting (petal in-rolling) of the flowers was delayed by continuously immersing the stems in a 5–20 mM PPOH solution. In addition, the continuous treatment with PPOH markedly reduced autocatalytic ethylene production of the petals accompanying senescence. This reduction of autocatalytic ethylene production was considered responsible for the inhibitory effect of PPOH on flower wilting. The inhibitory activity of trans-propenylphosphonic acid (trans-PPOH), on both flower wilting and the autocatalytic ethylene production accompanying senescence was markedly lower than that of PPOH, suggesting that PPOH action is stereoselective. PPOH may be of interest as a new, water-soluble inhibitor of wilting and autocatalytic ethylene production in cut carnation flowers.     

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.     

Ethanol reduces sensitivity to ethylene and delays petal senescence in cut Tweedia caerulea flowers

Tweedia caerulea flowers are sensitive to ethylene and the closing of the flowers, a characteristic of senescence, is accelerated by exposure to ethylene. T. caerulea flowers were continuously treated with ethanol at concentrations of 0, 2, 4, 6, 8, 10 or 12 %, and treatment levels at 4 % and above showed delayed closing. Ethanol accelerated climacteric increase in ethylene production from flowers. Although ethylene production was higher in gynoecium than in petals, ethanol treatment accelerated ethylene production by both organs. Exposure to ethylene increased autocatalytic ethylene production, and production was further accelerated by ethanol treatment. When flowers treated with ethanol were exposed to ethylene, senescence was delayed compared to that for untreated flowers, suggesting that ethanol reduces the sensitivity of flowers to ethylene. These results indicate that treatment with ethanol delays petal senescence in cut T. caerulea flowers, possibly through reduced sensitivity to ethylene.     

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     

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.     

Effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on carnation flower longevity

The effects of a novel preservative for cut carnation flowers, 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS), were investigated. DPSS extended the vase life of cut carnation flowers not only by continuous treatment but pulse treatment as well. This inhibition of senescence by DPSS appeared to depend on that of ethylene production in carnation flowers. DPSS provided no protection from the action of ethylene nor did it inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. It did inhibit ACC-dependent ethylene production in carnation petal discs, suggesting possible potential for inhibiting ACC oxidase.     

Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers

The Arabidopsis thaliana etr1-1 allele, capable of conferring ethylene insensitivity in a heterologous host, was introduced into transgenic carnation plants. This gene was expressed under control of either its own promoter, the constitutive CaMV 35S promoter or the flower-specific petunia FBP1 promoter. In about half of the transgenic plants obtained flower senescence was delayed by at least 6 days relative to control flowers, with a maximum delay of 16 days, a 3-fold increase in vase life. These flowers did not show the petal inrolling phenotype typical of ethylene-dependent carnation flower senescence. Instead, petals remained firm and finally started to rot and decolorize.In transgenic plants with delayed flower senescence, expression of the Arabidopsis etr1-1 gene was detectable and the expression pattern followed the activity of the upstream promoter. In these flowers expression of the ACO1 gene, encoding the final enzyme in the ethylene biosynthesis pathway, ACC oxidase, was down-regulated. This indicates that the autocatalytic induction of ethylene biosynthesis, required to initiate and regulate the flower senescence process, is absent in etr1-1 transgenic plants due to dominant ethylene insensitivity.The delay in senescence observed in transgenic etr1-1 flowers was longer than in flowers pretreated with chemicals that inhibit either ethylene biosynthesis (amino-oxyacetic acid) or the ethylene response (silver thiosulfate). This may have important implications for post-harvest management of carnation flowers.     

Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.     

Regulation of Senescence in Carnation (Dianthus caryophyllus): Effect of Abscisic Acid and Carbon Dioxide on Ethylene Production

Abscisic acid hastened senescence of carnation flowers and this was preceded by stimulation of accelerated ethylene production. Carbon dioxide delayed the onset of autocatalytic ethylene production in flowers regardless of treatment with abscisic acid. Flowers exhibited a low and transient climacteric of ethylene production without wilting while in 4% carbon dioxide and underwent accelerated ethylene production culminating in wilting when removed from carbon dioxide. Hypobaric ventilation, which lowers ethylene to hyponormal levels within tissues, extended flower longevity and largely negated enhancement of senescence by abscisic acid. Supplementing hypobarically ventilated flowers with ethylene hastened senescence irrespective of abscisic acid treatment. Collectively, the data indicate that abscisic acid hastens senescence of carnations largely as a result of advancing the onset of autocatalytic ethylene production.     

The mechanism involved in ethylene-enhanced ethylene synthesis in carnations

The plant hormone ethylene triggers and enhanced ethylene synthesis in certain ripening fruits and senescing flowers. Unlike most carnation (Dianthus caryophyllus L.) cultivars exhibiting climacteric rise in ethylene production at the onset of senescence, cv. Sandrosa does not show this phenomenon naturally. In order to understand the mechanism of autocatalytic ethylene production, we exposed carnation flowers cv. Sandrosa to ethylene which resulted in an enhanced capacity for ethylene synthesis in the petals. A short time response of one hour was measured for an increase in ACC oxidase activity, about five hours in advance of an increase in ACC synthase activity and ethylene production. The observed enhancement was dependent on the presence of exogeneous ethylene, and could be partially inhibited by prior treatment of the petals with -amanitin or cycloheximide. The results of the present study suggest that in response to ethylene, activation of an existing enzyme is taking place first. This is followed by an increase in expression of ACC oxidase and ACC synthase mRNAs.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DTT dithiothreitol - PMSF phenyl-methylsulfonyl fluoride - SAM S-adenosyl-L-methionine     

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.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Characteristics of the inhibitory action of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on ethylene production in carnation (Dianthus caryophyllus L.) flowers

1,1-Dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS)inhibited ethylene productionin carnation flowers during natural senescence, butdid not inhibit the ethyleneproduction induced by exogenous ethylene in carnationflowers, by indole-3-acetic acid (IAA) in mungbean hypocotylsegments and by wounding in winter squashmesocarp tissue. These findings suggested that DPSSdoes not directly inhibit ethylene biosynthesis fromL-methionine to ethylenevia S-adenosyl-L-methionine and1-aminocyclopropane-1-carboxylate. During naturalsenescence of carnation flowers, abscisic acid (ABA)was accumulated in the pistil and petals 2 days beforethe onset of ethylene production in the flower, andthe ABA content remained elevated until the onset ofethylene production. Application of exogenousABA to cut flowers from the cut stem end caused arapid increase in the ABA content in flower tissuesand promoted ethylene production in the flowers. These results were in agreement with the previousproposal that ABA plays a crucial role in theinduction of ethylene production during natural senescence incarnation flowers. DPSS preventedthe accumulation of ABA in both the pistil and petals,suggesting that DPSS exerted its inhibitory action onethylene production in naturally-senescing carnationflowers through the effect on the ABA-related process.     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine