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     

Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals

Senescence of carnation (Dianthus caryophyllus L. ev. White Sim) petals coincided with a decrease on a per flower basis in the yield of cell wall and ethanol-insoluble solids. The decrease in cell wall yield per flower was due largely to a loss of neutral sugars, primarily galactose (45%) and arabinose (23%). On a per flower basis, water-and chelator-soluble pectins increased throughout development, comprising in senescent petals 18 and 58%, respectively, of total pectin. Alkali-soluble pectins ranged from 35 to 45% of the total pectin and decreased during senescence. Gel chromatography of chelator- and alkali-soluble pectins revealed no change in molecular size and polygalacturonase activity was not detected. Large-molecular-size hemicelluloses decreased during development, an observation reminiscent of the changes affecting hemicelluloses during the ripening of a number of fruit types. Compositional analysis of the large hemicellulosic polymers revealed a decrease in xylose and galactose content.     

6-苄基腺嘌呤和激动素对香石竹切花衰老的生理效应

6-苄基腺嘌呤（6-BA）和激动素（KT）均能改善香石竹切花体内的水分平衡,增加切花的鲜重,增大花径,提高过氧化物酶（POD）活性,延缓可溶性蛋白质含量下降以及丙二醛（MDA）含量和O2^-·于生成速率的增加,延长切花瓶插寿命2～3d。     

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。     

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.     

Effect of oxygen concentration on the senescence and energy metabolism of cut carnation flowers

The effect of O₂ concentration on energy metabolism and senescence has been studied in cut flowers of Dianthus caryophyllus L. cv. Scania. As compared to the control (21% O₂), 5% O₂ delays flower senescence as well as decay of nucleotide level and AEC (adenylate energy charge). An atmosphere of 100% O₂ accelerates senescence as well as the decrease of nucleotide level and AEC. While anoxia brings about a faster decrease of ATP and AEC than of total nucleotides, hyperoxia brings about a faster decrease in adenyl nucleotides than in ATP and AEC values. Petal oxygen uptake is over 90% of the maximal value under 4% O₂ and saturates at 10% O₂. The development of senescence is dicussed as a two phase process (first phase-progressive and second phase-catastrophic) triggered by the action of hyperoxia, first on the system for energy utilization and later on the system for energy production, the degradation of which seems to be linked with increase in membrane permeability and withering.     

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.     

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.     

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.     

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.     

溴代十六烷基吡啶对香石竹切花的保鲜效应

研究切花瓶插液中加入0．3g．L^-1季铵盐化合物溴代十六烷基吡啶（CPB）后,瓶插液中微生物密度和切花茎杆基部病原菌分离频率下降,切花后期的水分平衡和鲜重能较好地维持,花冠直径增大,观赏寿命延长。     

Regulation of membrane phospholipid catabolism in senescing carnation flowers

Evidence has been obtained for the involvement of μ M levels of Ca²⁺ in phospholipid catabolism during petal senescence by following the breakdown of [U-¹⁴C]-phosphatidylcholine by microsomal membranes from cut carnation ( Dianthus caryophyllus L. cv. White-sim) flowers. Phospholipid degradation was mediated by three membrane-associated lipases, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4) and lipolytic acyl hydrolase. The activities of phospholipase D and phosphatidic acid phosphatase were stimulated by 30 and 100%, respectively, in the presence of 40 μ M free Ca²⁺, and the Ca²⁺-stimulation of phosphatidic acid phosphatase was calmodulin-dependent. When L-3-phosphatidyl-[2-³H]-inositol and L-3-phosphatidyl-[N-methyl-³H]-choline were used as substrates, inositol and choline accounted for 95 and 99%, respectively, of the water-soluble radiolabelled products. This suggests a predominance of phospholipase D activity over phospholipase C activity in these membranes.
Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre-labelled with ³²PO^3-₄ were treated with 10 ppm ethylene. All phospholipids incorporated the label, but no enhanced turnover of phosphatidylinositol was observed. Inositol 1,4,5-triphosphate did not release Ca²⁺ from preloaded microsomal vesicles at concentrations known to be effective in animal systems (i.e. < 5 μ M ) although release of Ca²⁺ was observed when a higher (20 μ M ) concentration was used.     

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.     

Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.)

Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.Abbreviations BAP 6-benzylaminopurine - Kinetin 6-furfurylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog     

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     

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.