Free radical production, catalase and superoxide dismutase activities and membrane integrity during senescence of petals of cut carnations (Dianthus caryophyllus)

There is a sudden increase in free radical levels, measured from the electron spin resonance (ESR) signal, in cut carnation ( Dianthus caryophyllus L. cv. Ember) petal powders between the end of blooming and the onset of withering. There is also an increase in the microsomal generation of superoxide radicals (measured from the ESR-Tiron signal). These increases correspond to a decrease of polar lipids content, a slight increase in peroxides and to the onset of a sudden efflux of electrolytes. A correlation is established between free radical production and the loss of membrane integrity. Catalase (EC 1.11.1.6) activity increases progressively until complete withering and an hypothesis concerning the action of this enzyme is proposed. The changes in superoxide dismutase (EC 1.15.1.1) activity appear to be independent of the amplitude of the ESR-Tiron signal.     

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.     

Changes in activities of superoxide dismutases during aging of petals of cut carnations (Dianthus caryophyllus)

Between the end of blooming and the withering of petals of cut carnations ( Dianthus caryophyllus L. cv. Ember), the total superoxide dismutase activity decreased. The three forms of superoxide dismutase (Cu, Zn-SOD, Fe-SOD and Mn-SOD. EC1. 15. 1. 1) identified on polyacrylamide gel exhibited several isozymes, the activities of which decreased sooner or later during aging. The important activity of the Mn-SOD decreased at the beginning of withering to become undetectable, while the weak activity of Fe-SODs declined only at the end of withering. It is suggested that the localization of SODs in the cell may explain these differences. Hypothesis are proposed to understand the decreased activity of SODs during aging.     

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.     

In vitro production of dianthin from crown gall lines of carnation (Dianthus caryophyllus L.)

Tumorous crown gall lines of carnation (Dianthus caryophyllus L.) were obtained by transforming leaf explants by the co-cultivation procedure; transformed lines were checked for their ability to produce in vitro the type 1 ribosome-inactivating proteins dianthins. Crude extracts from cultured callus were able to inhibit protein synthesis in a cell-free system based on rabbit reticulocyte lysate. A protein with an apparent molecular mass comparable to that of dianthin 30 from leaves was identified from its chromatographic behaviour and by Western analysis. Dianthin from callus showed a specific activity comparable to that reported for the leaf isoform. The presence and accumulation in the tumorous line of a 37 kDa protein with no ribosome-inactivating activity and strong antigenic reactivity to dianthins is discussed. Received: 18 July 1997 / Revision received: 10 January 1998 / Accepted: 5 February 1998     

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.     

Fungitoxic phenols from carnation (Dianthus caryophyllus) effective against Fusarium oxysporum f. sp. dianthi

The phenol compositions of two cultivars of carnation (Dianthus caryophyllus) namely "Gloriana" and "Roland", which are partially and highly resistant, respectively, to Fusarium oxysporum f. sp. dianthi have been investigated with the aim of determining if endogenous phenols could have an anti-fungal effect against the pathogen. Analyses were performed on healthy and F. oxysporum-inoculated in vitro tissues, and on in vivo plants. Two benzoic acid derivatives, protocatechuic acid (3,4-dihydroxybenzoic acid) and vanillic acid (4-hydroxy-3-methoxybenzoic acid), were found within healthy and inoculated tissues of both cultivars, together with the flavonol glycoside peltatoside (3-[6-O-(alpha-L-arabinopyranosyl)-beta-D-glucopyranosyl] quercetin). These molecules proved to be only slightly inhibitory towards the pathogen. 2,6-Dimethoxybenzoic acid was detected in small amounts only in the inoculated cultivar "Gloriana", while the highly resistant cultivar "Roland" showed the presence of the flavone datiscetin (3,5,7,2'-tetrahydroxyflavone). The latter compound exhibited an appreciable fungitoxic activity towards F. oxysporum f. sp. dianthi.     

Invertases of carnation petals. Partial purification, characterization and changes in activity during petal growth

Carnation ( Dianthus caryophyllus L. cv. White Sim) petals contained two distinct invertases (EC 3.2.1.26) based on chromatographic behavior on DEAE-cellulose. Both are soluble in 20 m M sodium phosphate buffer (pH 6.5) and exhibit acid pH optimum of 5.5. Extraction of a cell wall preparation from petals with 1 M NaCl released little additional activity. Furthermore, only traces of activity remained associated with the NaCl-extracted cell wall preparation. One of the soluble invertases, representing over 75% of the total activity, was partially purified by (NH₄)₂SO₄ fractionation and sequential chromatography over diethylaminoethyl-cellulose, concanavalin-A sepharose and polyacrylamide P-200. The enzyme was purified 38-fold with a recovery of 12%. It had an apparent native molecular weight of 215 kDa. The partially purified invertase is a β-fructofuranosidase (EC 3.2.1.26) based on its specificity for sucrose. The K_m for sucrose was 3.3 m M . Accumulation of reducing sugars and increased invertase activity during expansive petal growth indicates that sucrose is the major source of carbon for petal growth.     

Detection of 1-O-malylglucose: pelargonidin 3-O-glucose-6'-O-malyltransferase activity in carnation (Dianthus caryophyllus)

Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-β-d-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.     

Direct somatic embryogenesis and plant regeneration of carnation (Dianthus caryophyllus L.)

Conditions for efficient direct somatic embryogenesis and plant regeneration of leaf explants from carnation cultivars Lena (SIM group) and Bulgarian spray cultivars Nasslada, Yanita, Regina and Line 84 were established. Murashige and Skoog (MS) liquid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-benzylaminopurine was used for direct induction of embryoids without an additional callus phase. The first globular structures were observed after 20 days of cultivation. Their further development to the torpedo stage was correlated with the presence of polyethylene glycol (PEG 6000). Somatic embryo maturation was promoted by casein hydrolysate (1000 mg/l) in MS liquid media. The percentage conversion of embryos and polyembryos to whole plants varied between 10 and 75% among studied cultivars. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in a greenhouse. Received: 29 November 1996 / Revision received: 28 April 1997 / Accepted: 28 May 1998     

Flotation of lipid-protein particles containing triacylglycerol and phospholipid from the cytosol of carnation petals

Lipid-protein particles ranging from 20 to 250 nm in diameter have been isolated from the cytosol of carnation petals by flotation centrifugation and also by ultrafiltration. The cytosolic lipid-protein particles resemble oil bodies, lipid-protein particles found in oil-bearing seeds, in that they contain triacylglycerol, are circumscribed by phospholipid that is not organized in a bilayer, appear to be derived from membranes and can be isolated by flotation. However, the cytosolic particles are distinguishable from oil bodies in that triacylglycerol is not the dominant lipid. Indeed, they contain a spectrum of lipids in addition to phospholipids and triacylglycerol including free fatty acids, sterol and wax esters, phosphatidic acid and diacylglycerol. These same lipids are present in corresponding microsomal membranes as well, but in much smaller proportions relative to phospholipid. The lipid-protein particles from carnation petals contain a 17-kDa protein that is of similar size to oil body oleosin, but does not cross-react with anti-oleosin antibodies. The data indicate that these cytosolic particles are structurally and chemically similar to oil bodies and are consistent with the notion that their genesis may be a means of removing destabilizing lipids from membrane bilayers.     

RAPD and RFLP markers tightly linked to the locus controlling carnation (Dianthus caryophyllus) flower type

 Flower doubleness as a breeding characteristic is of major importance in carnation (Dianthus caryophyllus), one of the major cut-flowers sold worldwide, since flower architecture is of the utmost value in ornamentals. Based on the number of petals per flower, carnations are grouped into “single”, “semi-double” and “double” flower types. The first have five petals and are easily distinguishable, but of no economic value to the carnation industry. Flowers of standard and spray varieties, which constitute the largest market share, are usually of the double and semi-double type, respectively. These flower types are not easily distinguishable due to phenotypic overlaps caused by environmental conditions. To study the inheritance of this trait, several progeny segregating for flower type were prepared. Based on the number of single-flower type fullsibs among the offspring, we found that this phenotype is expressed only in plants homozygous for the recessive allele and that a dominant mutation in this allele causes an increase in petal number. Using random decamer primers, we identified a random amplified polymorphic DNA (RAPD) marker which is tightly linked to this recessive allele. The RAPD marker was cloned and used to generate a restriction fragment length polymorphic (RFLP) marker. This RFLP marker could discriminate with 100% accuracy between the semi-double and double- flower phenotypes in carnations of both Mediterranean and American groups. The advantages of RFLP over RAPD markers and their applicability to markerassisted selection in carnation are discussed. Received: 11 August 1997 / Accepted: 22 August 1997     

西花蓟马在康乃馨不同品种上的田间分布

调查西花蓟马Frankliniella occidentalis(Pergande)在10个康乃馨(Dianthus caryophyllus)品种上的发生与分布,比较花朵颜色与气味对西花蓟马寄主选择性的影响。结果表明,花朵上的西花蓟马数量在不同品种间存在显著差异。花朵颜色是影响西花蓟马寄主选择的主要原因,而花朵气味也能影响西花蓟马对寄主的选择性。     

Oxidative stress, antioxidant capacity and ethylene production during ageing of cut carnation (Dianthus caryophyllus) petals

The objective of this work was to study the role of free radicalsin relation to ethylene production during ageing of cut carnationpetals. Ethylene production by freshly cut flowers was negligible,but 8 d after cutting ethylene production began to increaseand reached a peak by day 9, before beginning to decline again.The efflux of electrolytes (membrane damage index) increased101% and 2',7'-dichlorofluorescein oxidation rate (oxidativestress index) increased 53% from day 8 to day 11 after detachment.Ethylene peak was either (a) not affected significantly by thesupplementation of exogenous ethylene on the day of cutting,or (b) expanded between days 7–9 after ethylene supplementationon day 6 of cutting, or (c) was inhibited by amino-oxyaceticacid and paraquat treatments. After ethylene supplementation,conductivity and 2',7'-dichlorofluorescein oxidation increasedsignificantly as compared to control petals, and the activityof antioxidant enzymes was not affected. However, both -tocopheroland glutathione content decreased significantly after ethylenesupplementation on day 6 after detachment. Amino-oxyacetic acidtreatment prevented the increases in conductivity and 2',7'-dichlorofluoresceinoxidation, did not alter the activities of antioxidant enzymesand significantly increased the content of -tocopherol and glutathioneas compared to control carnation petals. Paraquat treatmentparalleled qualitatively ethylene supplementation after 6 dof cutting. Taken as a whole, the data presented here may be understoodas experimental evidence of a close association between ethyleneproduction and oxidative stress in ageing of cut carnations. Key words: Carnation petals, oxidative stress, ethylene, antioxidants     

香石竹开花结实生物学研究

通过田间观察,运用TTC法、联苯胺-过氧化氢法、杂交指数、花粉-胚珠比和套袋实验等方法,对香石竹的开花状态及繁育系统进行了研究。结果表明:在人工栽培条件下,香石竹种群可周年开花,单株花期35～61d,单花花期9～15d;花粉在开花第1～5天具有活力,柱头在开花第4～12天具可授性,第8～11天可授性最强;杂交指数为5,P/O值为307.84～790.52,结合坐果率结果,判断其繁育系统属于异交,部分自交亲和,需要传粉者。     

1,1-Dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) does not inhibit the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase obtained from senescing carnation Dianthus caryophyllus L.) petals

The effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase isolated from senescing carnation petals were investigated. In contrast to a previous proposal, DPSS at 1 mM did not inhibit the in vitro activity of ACC oxidase. It was confirmed that DPSS does not inhibit ACC synthase activity. DPSS probably does not exert its inhibitory action on ethylene production by a direct action on ACC oxidase and ACC synthase, but by some unknown action.     

Effect of drought and prolonged refrigeration on senescence in cut carnation (Dianthus caryophyllus)

Severe water stress (40 and 50 h without water at 23°C) and long periods of refrigeration (4 and 5 weeks at 0°C) caused the peak of ethylene production by cut carnation flowers to appear soon after the return to normal conditions. Water stress caused a decrease in ψosm, but this increased back to the initial value on return to normal conditions. Accelerated wilting and massive ion leakage, probably a result of the Joss of membrane integrity, was associated with this premature burst of ethylene. Large amounts of acetaldehyde and ethanol accumulated during prolonged refrigeration (3, 4 or 5 weeks at 0°C). This accumulation of toxic metabolites may explain why the refrigeration of cut flowers for long periods causes a rapid wilting on return to normal conditions.     

Variants of carnation (Dianthus caryophyllus L.) obtained by organogenesis from irradiated petals

To obtain carnation variants differing from those produced by organogenesis alone, in vitro petal cultures were subjected to gamma irradiation. Histological analysis revealed the surface origin of buds and the different steps in meristem formation. A dose of 40 Gy administered on the fourth day of culture produced variants of horticultural interest in Niky. This period corresponded to dedifferentiation of cells that subsequently developed into bubs.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid