Carnation Petals Research Articles

G Protein Activation Stimulates Phospholipase D Signaling in Plants.

We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals.

Regulation of a petal‐specific ethylene‐induced 70‐kDa protein from Dianthus caryophyllus

Ethylene enhances senescence of carnation (Dianthus caryophyllus L.) flowers. Sephacryl S‐200 gel filtration followed by SDS‐PAGE of extracts of petals exposed to ethyiene revealed the increase or appearance of proteins with molecular masses of 70, 60, 35 and 33 kDa, and a concomitant decrease in proteins with molecular masses of 62. 45. 34. 30 and 26 kDa. The 70‐kDa protein, which was not detected in untreated flowers, was isolated and polyclonal antibodies were prepared against it. Its N‐terminal sequence (20 amino acids) was identical to that of a portion of the deduced protein encoded by cDNA clone pSR 12. recently isolated by others from carnation petals. Western blot analyses of petal extracts after various exposure times to ethylene alone or in the presence of translation or transcription inhibitors suggested that accumulation of the 70‐kDa protein is due primarily to ethylene‐induced synthesis. The apparent induction by ethyiene was further supported by data indicating that the protein is not formed in flowers pre‐treated with silver thiosulfale. an inhibitor of ethylene action, prior to exposure to ethylene. Ethylene induction of the protein was found only in petal tissue, and the protein was not detected in other carnation plant organs. Immunological studies revealed that the protein is specifically expressed in carnation petals and not in petals of several other flowers. Southern and northern analyses using an SRI2 cDNA probe indicated that the genomic DNA of petunia, carnation, and gerbera contain fragments homologous to SRI2. Gene expression products were however, only detected in petals of ethylenetreated carnation flowers.

Partial Characterization of Carnation Petal 1-Aminocyclopropane-1-Carboxylate Oxidase

Summary The plant hormone ethylene regulates senescence in carnation flowers. The final step in the biosynthesis of ethylene i.e. the conversion of ACC to ethylene is catalysed by the enzyme ACC oxidase. ACC oxidase was extracted from carnation petals and its characteristics were determined. The enzyme appears to be a soluble enzyme and requires Fe 2+ and ascorbate for maximal activity. CO 2 increases the reaction velocity and the apparent K m for ACC from 30 µM to 425 µM. Both Co 2+ and the ACC analog α-aminoisobutyric acid inhibit the ACC oxidase activity. The change in in vivo ACC oxidase activity in senescing carnation flowers is accompanied by a parallel change in ACC oxidase mRNA abundance and in vitro ACC oxidase activity. Optimal conditions for extraction and determination of authentic carnation petal ACC oxidase activity in vitro are described.

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

346 PRECURSORS OF SYSTEM II ETHYLENE SYNTHESIS IN SENESCING CARNATION PETALS

Methionine (MET) is considered the first committed precursor of ethylene (C2H4), and the pathway has been established as MET → S-adenosylmethionine (SAM) → 1-aminocyclopropane-1-carboxylic acid (ACC) → C2H4. It has been suggested that another pathway to C2H4 may exist, and this pathway has been labeled System II. Our objective was to evaluate several compounds as possible precursors of System II C2H4 production. `White Sim' carnations were placed continuously in 20 mM solutions of MET, ACC, δ-aminolevulinic acid, glutamic acid, α-ketoglutarate, or homocysteine. Deionized water was the control. C2H4 production from the entire flower was measured, and ACC in the basal portion of the petals was quantified. Flowers treated with ACC exhibited the greatest C2H4 production and accumulation of ACC. Homocysteine caused greater production of C2H4 and accumulated more ACC than MET and the other possible precursors. These results suggest that homocysteine may be involved in System II C2H4 production in senescing carnation petals.

Age-Related Changes in Biochemical and Physical Properties of Carnation Petal Plasma Membranes

Summary Senescence of flower petals, as of other plant organs, is accompanied by numerous well-defined compositional, structural and functional changes in their cellular membranes. The aim of this work was to compare the modifications occurring in the bulk of the cellular membranes, the microsomal fraction, to those occurring in a purified plasma membrane preparation. Microsomal membranes were prepared from petals of carnation ( Dianthus caryophyllus L., cv. White Sim) using differential centrifugation, plasma membranes were isolated from this fraction using two-phase partition. Of the various membrane marker enzymes tested, vanadate-sensitive ATPase showed the most pronounced decline with age. Proteins and phospholipids declined in both microsomal and plasma membranes. In contrast, free sterol content did not change with age in microsomal membranes but declined in plasma membranes. Membrane lipid fluidity, assessed through fluorescence polarization of 1,6-diphenyl hexatriene, was lower in plasma membranes and decreased with age in both membrane preparations. The binding of GTP to microsomal membranes declined significantly with age. In contrast, binding of GTP to plasma membranes was similar to GTP binding to microsomal membranes from young petals and did not change with age. Use of the thiol specific fluorescent probe, N-dansylaziridine, showed that membrane protein thiols declined quantitatively with age in both microsomal and plasma membranes. However, while the two preparations differed with respect to the labeling rates, the similar fluorescence spectra of the labeled preparations suggests that the polarities of the protein thiols in the two membrane types are comparable. We conclude that microsomal membranes may represent the petal plasma membranes in senescence-related studies of most properties, but not in that of GTP binding or kinetics of protein thiol labeling.

Recovering vitrified carnation (Dianthus caryophyllus L.) shoots using Bacto-Peptone and its subfractions

Vitrified shoots regenerated from carnation petals (Dianthus caryophyllus L. cv. Scania) were recovered by culturing them in a medium containing 3.0 g/l Bacto-Peptone. Wax structures not found on vitrified shoots developed on the abaxial surface of leaves of recovered shoots and on those of normal leaves. Recovered shoots were rooted and successfully acclimatized while vitrified shoots could not survive the acclimatization process. The Bacto-Peptone solution was fractionated and the efficiency of each fraction for the recovery of vitrification was examined. Only basic, non high molecular fractions whose molecular weight was less than 10,000 were effective.

An efficient method for adventitious shoot regeneration from cultured carnation petals

A highly efficient procedure for adventitious shoot regeneration from various parts of carnation petals using liquid media was developed. In contrast to a standard agar-containing medium, in which shoots regenerated from about half of the petals, on the liquid medium shoot regeneration was obtained from nearly all the petals. These shoots regenerated from various parts of the petal, and up to seven adventitious shoots were obtained per petal in cultivar ‘White Sim’, as well as in other carnation cultivars. The adventitious origin of the regenerated shoots was confirmed by scanning electron microscopy. The use of a liquid media did not affect the extent of vitrification, it being similar to that observed on agar.

Isolation of a ripening and wound-induced cDNA from Cucumis melo L. encoding a protein with homology to the ethylene-forming enzyme.

A cDNA clone (pMEL1) was isolated from a climacteric melon fruit cDNA library using the tomato ethylene-forming-enzyme (EFE) cDNA, pTOM13, as a probe. Northern analysis of RNA isolated from wounded leaf and fruit tissue and from preclimacteric and climacteric pericarp tissue was used to determine the pattern of pMEL1 RNA expression. pMEL1 hybridized to a 1.3-kb transcript in climacteric fruit and wounded leaf tissue but was undetectable in preclimacteric fruit and unwounded leaves. Maximal expression of pMEL1 RNA occurred in wounded ripe fruit. pMEL1 contained a 1230-bp insert containing a predicted open reading frame of 318 amino acids and molecular mass of 35.3 kDa. The predicted amino acid sequence of pMEL1 was 73-81% identical to the deduced amino acid sequences of tomato (pTOM13) EFE and EFE-related genes isolated from tomato and avocado fruit and senescent carnation petals. Genomic Southern analysis indicated that pMEL1 hybridized to a number of genomic fragments consistent with the presence of more than one pMEL1-related gene in melon. On Western analysis of total protein extracts from ripe tomato and melon fruit, using an antibody raised against tomato EFE (pTOM13) expressed in Escherichia coli, a single 35.5-kDa protein was detected. A 35-kDa product translated from in-vitro transcribed pMEL1 and immunoadsorbed by anti-EFE serum was very similar in size to the predicted 35.3-kDa pMEL1 cDNA protein product. These results indicate that pMEL1 may encode an EFE gene involved in ethylene biosynthesis during fruit ripening and may be identical to or share extensive sequence similarity with an EFE expressed in response to tissue wounding.

THE EFFECT OF POLYAMINES ON THE SENESCENCE OF CARNATION PETALS.

The interaction between polyamines and ethylene is still not clear for floral tissues. The aim of the present paper is to examine the senescence on the isolated petals of carnation (Dianthus caryophyllus cv. Desio) but not the whole flower in an attempt to clarify the exact role of polyamines. Petals were treated with putrescine (Put; 0.0, 1.0, 10mM), spermidine (Spd; 0.0, 1.0, 10mM), spermine (Spn; 0.0, 1.0, 10mM), Put+Spd (1.0mM), Put+Spn (1.0mM). The fresh weight of petals in all 10mM treatment was decreased less than those in the other treatments at all times but there were no significant differences. The differences in ethylene production were significant. In petals maintained in 10mM of polyamines, ethylene production was completely inhibited until 13 days and senescence was considerably retarded. However, ethylene productions in 1.0mM polyamines treatments were delayed 2-3 days with reduced amounts. These results suggest that high concentrations of polyamines retard senescence and completely inhibit ethylene production. ACC content, activities of ACC synthase and SAM decarboxylase were analyzed. Finally, the role of SAM in ethylene and polyamines biosynthesis will be discussed.

Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents.

In situmicro-spectrophotometric and micro- spectrocolorimetric investigation of vacuolar pigments in flowers of cultivars of carnation(.Dianthus caryophyllus)

Living epidermal cells of flower petals of carnations have been surveyed for their native colour by in situ spectral (350-750 nm wavelengths) and colorimetric investigations (CIE- Lab, L* C* h system) of their vacuolar pigment content with a micro-spectrophotometer. Vacuolar solutions in cultivars with yellow based colours generally display high Absorbances (close to, or over A = 2 under a 25 |xm optical depth) in the near-UV area only: at 360-375 nm for ivory or pale yellow colours (flavonols only) and additionally at 380- 395 nm (chalcone glycosides) for yellow ones. Carnation cultivars with cyanic colours are accumulating anthocyanins, namely monosides and diosides of pelargonidin and cyani- din, along with flavonol glycosides acting as co-pigments. Besides their main A.max in the 520-550 nm area, frequently appearing at lower wavelengths than in those of diosides, spectra of solutions of monosides are characterized by an additional peak or shoulder around 450 nm. Cyanidin derivates often exhibit highe...

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.

Characterization of an ethylene-regulated flower senescence-related gene from carnation

The programmed senescence of carnation (Dianthus caryophyllus L.) petals requires active gene expression and is associated with the expression of several senescence-related mRNAs. Expression of the mRNA represented by the cDNA clone pSR12 has previously been shown to be transcriptionally activated by ethylene specifically in senescing flowers. We report in this paper the structural analysis of this cDNA and its corresponding gene. One cloned genomic DNA fragment, SR12-B, contained the entire transcription unit in 17 exons, interrupted by 16 introns. A second gene, SR12-A, was highly homologous to SR12-B with several nucleotide substitutions and a 489 bp deletion in the 5' flanking DNA sequence. The SR12 transcript has an open reading frame of 2193 bp sufficient to encode a protein of 82.8 kDa. No significant homology at the DNA or protein levels was found with other known genes. We have identified a DNA-binding factor which specifically interacts with two upstream fragments (-149 to -337 and -688 to -1055) of SR12-B. Both fragments apparently compete for the same binding factor. The DNA-binding activity was present in nuclear extracts from both presenescent and senescing carnation petals. The upstream DNA fragments that bind this factor have sequence homology with promoter sequences of other ethylene-regulated genes.

Role of the Gynoecium in Cytokinin-induced Carnation Petal Senescence

Treatment of cut carnation (Dianthus caryophyllus L. `White Sim') flowers with the synthetic cytokinin benzyladenine (BA) at concentrations >1.0 μm induced premature petal senescence. Flowers treated with 100 μm BA exhibited elevated ethylene production in styles and petals before untreated flowers. The gynoecia of BA-treated flowers accumulated 1-aminocyclopropane-l-carboxyllc acid (ACC) and enlarged before untreated flowers. Removal of the gynoecium (ovary and styles) or styles prevented BA-induced petal senescence and resulted in a substantial delay in petal senescence. In contrast, removal of the gynoecium had no effect on timing of petal senescence in flowers held in water. These results indicate BA stimulates petal senescence by inducing premature ACC accumulation and ethylene production in the gynoecium.

Endoglycanase-Catalyzed Degradation of Hemicelluloses during Development of Carnation (Dianthus caryophyllus L.) Petals

Large molecular-size hemicelluloses, including xyloglucan, decreased in quantity during development of carnation (Dianthus caryophyllus L. cv White Sim) petals, along with a relative increase in polymers with an average size of 10 kilodaltons. An enzyme extract from senescing petal tissue depolymerized the large molecular-size hemicelluloses in a pattern similar to that occurring in vivo during petal development. The products generated in vitro were composed of polymeric and monomeric components, the latter consisting primarily of xylose, galactose, and glucose. The 10 kilodalton hemicelluloses were resistant to in vitro enzymic hydrolysis. Glycosyl-linkage composition of the large molecular-size polymers provided evidence for the presence of xyloglucan with smaller amounts of arabinoxylan and arabinan. The 10 kilodalton polymers were enriched in mannosyl and 4-linked glucosyl residues, presumably derived from glucomannan. During petal development or enzymic hydrolysis, no change was observed in the relative glycosyl-linkage composition of the large molecular-size hemicelluloses. The in vitro activity of carnation petal enzymes active toward native hemicelluloses increased threefold at the onset of senescence and declined slightly thereafter. Gel chromatography revealed 23 and 12 kilodalton proteins with hemicellulase activity. The enzymes hydrolyzed the large molecular-size hemicelluloses extensively and without formation of monomers. Endoxylanase activity was detected in the partially purified enzyme preparation. Xyloglucan was depolymerized in the absence of cellulase activity, suggesting the presence of a xyloglucan-specific glucanase. These data indicate that the hemicellulose molecular-size changes observed during development of carnation petals are due, in part, to the enzymic depolymerization of large molecular-size hemicelluloses.

Acyl Chain and Head Group Regulation of Phospholipid Catabolism in Senescing Carnation Flowers

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.

Distinguishable patterns of phospholipid susceptibility to catabolism in senescing carnation petals

Microsomal membranes isolated from petals of senescing carnation petals are able to enzymatically degrade exogenous radiolabelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. A comparison of the rates of catabolism for various radiolabelled molecular species of each of these phospholipid classes suggests that microsomal phospholipids have different susceptibilities to degradation. The same degradation preferences are exhibited by young and senescent membranes, and they appear to reflect recognition by the degrading enzyme of both the head group and acyl chain composition of the phospholipid. There is no increase with advancing senescence in the capability of isolated microsomal membranes to catabolize radiolabelled phospholipids, and there is no change with age in phospholipid composition of microsomal membranes. Yet, levels of microsomal phospholipid decline with advancing senescence by ca three-fold relative to corresponding levels of membrane free sterols. The data indicate that the decline in microsomal phospholipid with advancing petal senescence cannot be attributed to increased membrane-associated phospholipase activity, and they raise the possibility that provision of particular molecular species of phospholipids with increased susceptibility to degradation could be the underlying basis for enhanced phospholipid catabolism during petal senescence.

Isozymes of Superoxide Dismutase in Mitochondria and Peroxisomes Isolated from Petals of Carnation (Dianthus caryophyllus) during Senescence

The balance between reactions involving free radicals and processes which ameliorate their effect plays an important role in the regulation of plant senescence. In this study a method was developed to isolate peroxisomes and mitochondria from carnation (Dianthus caryophyllus L. cv Ember) petals. Based on electron microscopy and marker enzyme levels, the proportion of peroxisomes to mitochondria increases during senescence. The superoxide dismutase (SOD) content of these fractions was examined. Mitochondria and peroxisomes were shown to contain two electrophoretically distinct SODs, a manganese-, and an ironcontaining SOD. The Mn- and Fe-SOD were found to have relative molecular weights of 75,000 and 48,000 and isoelectric points of 4.85 and 5.00, respectively. The presence of a Fe-SOD in mitochondria and peroxisomes is unique because this enzyme is usually located in chloroplasts. The activity of these two isoenzymes decreased during senescence in mitochondria but remained high in peroxisomes from senescent tissue. It is suggested that peroxisomes play a particular role in the process of senescence.

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.