Senescence Of Carnation Flowers Research Articles

DNA methylation remodeled amino acids biosynthesis regulates flower senescence in carnation (Dianthus caryophyllus).

Dynamic DNA methylation regulatory networks are involved in many biological processes. However, how DNA methylation patterns change during flower senescence and their relevance with gene expression and related molecular mechanism remain largely unknown. Here, we used whole genome bisulfite sequencing to reveal a significant increase of DNA methylation in the promoter region of genes during natural and ethylene-induced flower senescence in carnation (Dianthus caryophyllus L.), which was correlated with decreased expression of DNA demethylase gene DcROS1. Silencing of DcROS1 accelerated while overexpression of DcROS1 delayed carnation flower senescence. Moreover, among the hypermethylated differentially expressed genes during flower senescence, we identified two amino acid biosynthesis genes, DcCARA and DcDHAD, with increased DNA methylation and reduced expression in DcROS1 silenced petals, and decreased DNA methylation and increased expression in DcROS1 overexpression petals, accompanied by decreased or increased amino acids content. Silencing of DcCARA and DcDHAD accelerates carnation flower senescence. We further showed that adding corresponding amino acids could largely rescue the senescence phenotype of DcROS1, DcCARA and DcDHAD silenced plants. Our study not only demonstrates an essential role of DcROS1-mediated remodeling of DNA methylation in flower senescence but also unravels a novel epigenetic regulatory mechanism underlying DNA methylation and amino acid biosynthesis during flower senescence.

Differential regulation of two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes, including the additionally cloned DcACO2, during senescence in carnation flowers

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) catalyzes the final step of ethylene biosynthesis. ACO proteins are also reportedly encoded by a multigene family. Although the presence of several ACO genes is suggested in carnation (Dianthus caryophyllus L.), DcACO1 is the only ACO gene in which full-length cDNA has been isolated. This study aimed to clone another ACO gene in carnation flowers and investigate changes the expression of the two ACO genes in floral organs during flower senescence. We cloned a homolog of the ACO gene from carnation gynoecium and designated it DcACO2. Transcript levels of DcACO2 were higher in the style and ovary than in the petals at harvest, although DcACO2 transcript levels in these organs increased during flower senescence. The ACO activity in the style was very high at harvest, suggesting that this high activity could be attributed to the translation of the DcACO2 transcript. However, DcACO2 transcript was only detected at very low levels in the petals of senesced flowers. Ethylene and ACC treatments accelerated petal wilting and increased ethylene production of the petals, style, and ovary. In the style and ovary, the DcACO1 transcript level was markedly higher than the DcACO2 transcript level as a result of ethylene and ACC treatments, and DcACO1 transcript levels in the petals were also increased by ethylene and ACC treatments. These results indicate that the expression of DcACO1 and DcACO2 are differently regulated among floral organs during senescence in carnation flowers.

Blue light postpones senescence of carnation flowers through regulation of ethylene and abscisic acid pathway-related genes

Endogenous signals in response to exogenous factors determine the senescence of flowers. Interactions among phytohormones especially abscisic acid (ABA) and ethylene are the major determinant of the senescence. In the present study, complex expression patterns of the genes related to ABA and ethylene as endogenous signals were investigated on cut carnations (Dianthus caryophyllus L.) that were exposed to different light spectra. Expression of ethylene biosynthetic (DcACS and DcACO), and signaling (DcETR and DcEin2) genes and also genes involved in ABA biosynthesis (DcZEP1 and DcNCED1), transport (DcABCG25 and DcABCG40) and catabolism (DcCYP707A1) were evaluated in petals of carnations exposed to three light spectra [white, blue and red]. Lowest relative membrane permeability (RMP) was detected in flowers that exposed to Blue light (BLFs), as a consequence, the longest vase life was found in BLFs. The Red and White lights markedly accelerated flower senescence and increased expression of DcACS and DcACO on day 6 and 10 of vase life assessment respectively; while Blue light inhibited the expression of ethylene biosynthetic genes. Expression of the genes involved in the production and transport of ABA and in signal transduction of ethylene was elevated during vase life of flowers irrespective of exposure to different light spectra. In conclusion, Blue light can be an effective environmental factor to extend the vase life of carnation flowers by delaying the petal senescence through down-regulation of ethylene biosynthetic genes and up-regulation of ABA biosynthetic genes.

Flowering profile and ethylene production of a new carnation subspecies 'Mini-tiara'

Ethylene is involved in the senescence of carnation flowers. It is synthesized abundantly after full opening of the flowers and accelerates wilting of petals, which results in withering of the flowers. We investigated the possible involvement of ethylene production in the loss of display value of cut flowers of ‘Mini-tiara’ carnation, a new subspecies derived from Dianthus caryophyllus L. by interspecific cross with wild-type Dianthus species (wild pinks). Flowers of ‘Mini-tiara’ carnation have a unique morphology with pointed-shaped petals, some of which in the middle part of the flowers stand straight and build a dome throughout the display time, and lose their display value when the dome collapses by bending all petals outside (full opening of flowers). Ethylene evolution from ‘Mini-tiara’ carnation was not detected from flowers with upstanding petals, but occurred several days after the collapse of the dome (after full opening of the flowers), the time they already lost their display value. These findings indicated that ethylene production is not engaged in the loss of display value of ‘Mini-tiara’ carnation.

Ethylene production associated with petal senescence in carnation flowers is induced irrespective of the gynoecium

To clarify whether climacteric-like increases in ethylene production of senescing petals are also induced in the absence of the gynoecium in cut carnation (Dianthus caryophyllus cv. Barbara) flowers, we compared ethylene production and expression of ethylene-biosynthesis genes in detached petals and in petals, which remained on flowers (attached petals). No significant difference in longevity was observed between the attached and detached petals when held in distilled water, and both showed the inward rolling typical of senescing flowers. Treatment with silver thiosulfate complex (STS), an ethylene inhibitor, similarly delayed senescence of attached and detached petals. Climacteric-like increases in ethylene production of petals and gynoecium started on the same day, with similar bursts in attached and detached petals. Transcript levels of DcACS1 and DcACO1 were very low at harvest and increased similarly during senescence in both petal groups. Removal of the gynoecium did not significantly delay wilting of attached petals. In flowers with the gynoecium removed, the petals produced most of the ethylene while production by the other floral organs was very low, suggesting that wound-induced ethylene is not the reason for the ineffectiveness of gynoecium-removal in inhibiting flower senescence. These results indicate that ethylene biosynthesis is induced in carnation petals irrespective of the gynoecium.

Role of ABA in Triggering Ethylene Production in the Gynoecium of Senescing Carnation Flowers: Changes in ABA Content and Expression of Genes for ABA Biosynthesis and Action

In senescing carnation (Dianthus caryophyllus L.) flowers, ethylene production begins in the gynoecium, and the resulting ethylene acts on petals, inducing autocatalytic ethylene production. We investigated the role of abscisic acid (ABA) in ethylene production in the gynoecium of flowers. First, cDNAs of major genes involved in ABA biosynthesis and signaling were cloned from carnation flower tissues. Then, changes in ABA content and gene expression of ABA biosynthesis and signaling in the ovary were examined using three cultivars, ‘Light Pink Barbara (LPB)’ and ‘Excerea’, whose cut flowers produce ethylene during senescence and have an ordinary vase-life of about one week, and ‘Miracle Rouge’, whose cut flowers produce no detectable ethylene during senescence and have a vase-life of about three weeks. ABA content in the ovary was 530–710 pmol·g−1 fresh weight (FW) from Os 2 (early opening stage) to Os 6 (end of opening stage) in ‘LPB’, and at 200–380 pmol·g−1 FW in ‘Excerea’ at the same stages; but 930 pmol·g−1 FW at Ss 1 (early senescence stage). The ABA content remained at 70– 160 pmol·g−1 FW in ‘Miracle Rouge’. The changes in ABA content were in parallel with the transcript levels of DcNCED1 (carnation gene for 9-cis-epoxycarotenoid dioxygenase). DcPYR1 (ABA receptor gene) transcript was 0.004–0.007 relative expression level (r.e.l.) in ‘LPB’ ovary at Os 1–Os 3, and 0.028 r.e.l. at Ss 1 (beginning of senescence stage). In ‘Excerea’ ovary, DcPYR1 transcript was 0.025–0.037 r.e.l. during flower opening and higher at Ss 1. By contrast, DcPYR1 transcript remained at 0.002–0.006 r.e.l. in ‘Miracle Rouge’ ovary during flower opening and senescence. The transcripts of DcACS1, the key gene for ethylene biosynthesis, were detected at Ss 1 in ‘LPB’, and at Ss 2 in ‘Excerea’, but not in ‘Miracle Rouge’ throughout flower opening and senescence stages. These findings suggest that ABA plays a causal role in inducing the expression of the DcACS1 gene in the gynoecium, leading to ethylene biosynthesis, and that both the ABA content and DcPYR1 expression must be above putative threshold levels for ABA to exert its action.

Ethylene Production and Petal Wilting during Senescence of Cut Carnation (Dianthus caryophyllus) Flowers and Prolonging Their Vase Life by Genetic Transformation

Senescence of carnation flowers is characterized by autocatalytic ethylene production from petals and subsequent wilting of the petals. Recent studies on the regulation of ethylene production and wilting in senescing carnation petals revealed that (1) petal senescence is triggered by ethylene evolved from the gynoecium during natural senescence, (2) ethylene production in the gynoecium is induced by a factor(s) other than pollination signals in carnation flowers lacking anthers, (3) there are two subsets of ethylene responses in the petals, one responsible for autocatalytic ethylene production and the other for wilting, (4) expression of genes involved in the execution of petal withering is differently regulated between ethylene-dependent or -independent senescence. Furthermore, it was revealed that the generation of transgenic carnation without detectable ethylene production is useful to prolong the vase life of cut carnation flowers.

Repressed Expression of DC-ACS1 Gene in a Transgenic Carnation Supports the Role of its Expression in the Gynoecium for Ethylene Production in Senescing Flower

DC-ACS1 is a gene for 1-aminocyclopropane-1-carboxylate (ACC) synthase which is expressed slightly in the gynoecium, but abundantly in petals of carnation (Dianthus caryophyllus L.) flowers. It is known that ethylene evolved from the gynoecium triggers the expression of DC-ACS1 and DC-ACO1 (the gene for carnation ACC oxidase), which results in an abundant ethylene production in petals. In a transgenic carnation (16-0-66 line) transformed with DC-ACS1 cDNA in sense orientation, the exogenous ethylene treatment caused the expression of DC-ACO1 in the gynoecium and petals, but not that of DC-ACS1. This suggests that the cosuppression of the expression of endogenous DC-ACS1 gene by the integrated transgene. DC-ACS1 transcript is absent in both gynoecium and petals of senescing 16-0-66 flowers, so that the flowers produce no ethylene; whereas DC-ACO1 transcript is present in the gynoecium but absent in the petals. These findings give further support on the role of the DC-ACS1 gene in the gynoecium for ethylene production by petals in senescing carnation flowers.

Cloning and Characterization of a cDNA Encoding a Putative Nuclease Related to Petal Senescence in Carnation (Dianthus caryophyllus L.) Flowers

Petal wilting, which involves the degradation of cell constituents, such as proteins, polysaccharides, lipids, and nucleic acids, is a determinant of the vase-life of carnation (Dianthus caryophyllus L.) flowers. In this study, a cDNA fragment, encoding a putative nuclease (DcNUC1), was isolated from carnation petals. In the petals of senescing carnation flowers, the level of the DcNUC1 transcript increased, corresponding to the increase of ethylene production. It became maximal in wilted flowers, which suggested the association of DcNUC1 expression with the wilting in carnation flowers.

MECHANISM OF SENESCENCE IN CARNATION FLOWERS

Senescence of carnation flowers is characterized by autocatalytic ethylene production from petals and subsequent wilting of the petals. Recent studies on the regulation of ethylene production and wilting in senescing carnation flowers revealed that (1) petal senescence is triggered by ethylene produced from the gynoecium during natural senescence, (2) there are two subsets of ethylene responses in the petals; one responsible for autocatalytic ethylene production and the other for wilting, and (3) in the petals ACC oxidase (DC-ACO1) and ACC synthase (DC-ACS1) genes are sequentially in this order. The expression of the DC-ACO1 gene probably affects that of the DC-ACS1 gene.

TOWARDS UNDERSTANDING THE ONSET OF PETAL SENESCENCE: ANALYSIS OF ETHYLENE PRODUCTION IN THE LONG-LASTING CARNATION CV. WHITE CANDLE

In senescing carnation (Dianthus caryophyllus L.) flowers, ethylene is produced from the gynoecium, and acts as a diffusible signal received by petals to induce the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase (DC-ACS1) and ACC oxidase (DC-ACO1) genes in the petals. This results in autocatalytic ethylene production in the petals. We investigated ethylene production in cut flowers of cv. White Candle, which produce ethylene only in trace amounts and have a long vase life. The low ethylene production in these flowers was due to low ethylene production in the gynoecium, accompanied by low accumulation of ACC synthase transcripts. These findings further support the importance of ethylene production from the gynoecium in the senescence of carnation flowers. Cv. White Candle flowers had water relations that were different from those in cv. Light Pink Barbara, a cultivar that showed the normal climacteric rise of ethylene production during senescence. We discuss a factor that possibly induces ethylene production in the carnation gynoecium.

Repressed ethylene production in the gynoecium of long-lasting flowers of the carnation 'White Candle': role of the gynoecium in carnation flower senescence.

Ethylene production and expression of ethylene biosynthetic genes was investigated in senescing flowers of carnation (Dianthus caryophyllus L.) cultivars 'White Candle (WC)' and 'Light Pink Barbara (LPB)', with long and short vase-lives, respectively. Ethylene production from the gynoecium and petals of senescing 'WC' flowers was below the limit of detection, in agreement with the repressed ethylene production from the whole flowers. However, exogenous ethylene treatment caused the accumulation of transcripts for DC-ACS1 and DC-ACO1 genes in both the gynoecium and petals, resulting in ethylene production from the flowers. Moreover, application of ABA or IAA, which are known to exhibit their action through the induction of ethylene synthesis in the gynoecium, to 'WC' flowers from their cut stem-end induced ethylene production and wilting in the flowers. These findings suggested that, in 'WC' flowers the mechanism of ethylene biosynthesis, i.e. the induction of expression of genes for ethylene biosynthesis and the action of resulting enzymes, was not defective, but that its function was repressed during natural senescence. Transcripts of DC-ACO1, DC-ACS3, and DC-ACS1 were present in the gynoecium of senescing 'LPB' flowers. In the gynoecium of senescing 'WC' flowers, however, the DC-ACO1 transcript was present, but the DC-ACS1 transcript was absent and the DC-ACS3 transcript was detected only in a small amount; the latter two were associated with the low rate of ethylene production in the gynoecium of 'WC' flowers. These findings indicated that the repressed ethylene production in 'WC' flowers during natural senescence is caused by the repressed ethylene production in the gynoecium, giving further support for the role of the gynoecium in regulating petal senescence in carnation flowers.

Senescence and Gene Expression of Transgenic Non-ethylene-producing Carnation Flowers.

Cut flowers of a transgenic carnation (Dianthus caryophyllus L.) line (sACO-1 line), which was transformed with a carnation 1-aminocyclopropane-1-carboxylate (ACC) oxidase cDNA in sense orientation (sACO transgene), had a longer vase life than had flowers of the non-transformed plant and produced only a negligible amount of ethylene during natural senescence. The suppressed ethylene production in sACO-1 line was not accompanied with the accumulation of mRNA for ACC oxidase (DC-ACO1) gene in the gynoecium or mRNAs for DC-ACO1 and ACC synthase (DC-ACS1) genes in the petals. Previously, it was shown that in naturally senescing carnation flowers, ethylene is first produced from the gynoecium ; the evolved ethylene, acting as a diffusible signal which is perceived by petals, induces the expression of DC-ACO1 and DC-ACS1 genes that results in autocatalytic ethylene production in the petals (Shibuya et al., 2000). The results indicate that sACO transgene inhibits the expression of DC-ACO1, probably by cosuppression in the gynoecium, which then suppresses ethylene production in all flowers of sACO-1 line.

Preservative solutions containing boric acid delay senescence of carnation flowers

We investigated the effect of a preservative solution containing boric acid on the senescence of cut carnation flowers (Dianthus caryophyllus L. cv. Master). A 24-h pulse treatment with the preservative solution containing 50, 75 or 100 mM boric acid or continuous treatment with 1 mM boric acid resulted in strong inhibition of the climacteric ethylene production. Both the pulse and continuous treatments significantly increased flower longevity. Free and conjugated 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase activity increased in carnation petals during senescence, although significantly less in boric acid-treated carnations than in control flowers. The levels of putrescine increased as senescence progressed in both control and boric acid-treated carnations and an increase in spermidine levels was higher in treated carnations. Abscisic acid levels in petals also increased during senescence, but much less in boric acid-treated carnations. It is concluded that boric acid prevents the early rise in ethylene production and considerably improves carnation vase life.

Fragrance volatiles of developing and senescing carnation flowers

Thirteen major volatiles of the carnation flower fragrance signature have been identified by GC/MS. Of these, ten, hexanal, (2E)-hexenal, 1-hexanol, 2-hexanol, 3-hexen-1-ol, nonanal, benzaldehyde, benzyl alcohol, benzyl benzoate and caryophyllene, were quantified. The steady-state levels of these ten volatiles change independently as the flowers develop and senesce, suggesting that their synthesis is developmentally regulated. In addition, the chemical composition of the fragrance signature in naturally senesced flowers proved to be very different from that for flowers that had been induced to senesce prematurely by treatment with ethylene. Thus, senescence-related changes in carnation floral scent appear not to be directly regulated by ethylene. From cellular fractionation studies, it is evident that all of the volatiles, except 2-hexanol, are present in both membranous and cytosolic compartments, suggesting that their synthesis is membrane-associated and that they subsequently partition into the cytosol in accordance with partition coefficients.

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.

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.

Is Acetaldehyde the Causal Agent in the Retardation of Carnation Flower Senescence by Ethanol?

Summary A 3 % ethanol holding solution inhibits the conversion of 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene, and doubles the vase-life of cut carnation flowers. Although the most effective concentration varies during experimentation, high concentrations of ethanol have negative effects. The most pronounced of these is the death of the ovary. Ethanol levels are much higher in the ovaries of treated flowers, than control flowers. A large amount of acetaldehyde was detected in the ovaries of treated flowers. Acetaldehyde was also detected in all other floral organs tested. Acetaldehyde is present in the head-space surrounding ethanol treated flowers, but not control flowers. This indicates that some of the ethanol applied as a post-harvest treatment is broken down to acetaldehyde, within the plant tissue, and released into the micro-atmosphere by the flower. If the conversion of ethanol to acetaldehyde is impeded, using an inhibitor of alcohol dehydrogenase, 4-methyl pyrazole, ethanol is no longer effective. Acetaldehyde, (1 and 3 %), as a holding solution, gave similar results as a 3 % ethanol holding solution. It appears that acetaldehyde, produced from ethanol, is the causal agent for the retardation of carnation flower senescence.

The use of acetaldehyde to control carnation flower longevity

Acetaldehyde is the causal agent of ethanol-induced longevity increases in carnation cut flowers. It increases the vase life of cut carnation flowers by at least 50%. The capacity of acetaldehyde to regulate carnation flower senescence was therefore investigated. Ethylene formation was reduced or inhibited as a result of acetaldehyde application. There was, however, no prevention of ethylene action. The morphological development of the ovary was also inhibited, thus eliminating the movement of metabolites from the petals. The potential use of acetaldehyde as a post-harvest treatment is however impractical, due to the inefficiency of pulse treatments and ineffectiveness in preventing the action of exogenous ethylene.

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.