Transient Water Stress in Carnation Flowers: Effect of Amino-oxyacetic Acid

A short and temporary water stress imposed on cut carnationflowers (Dianthus caryophyllus L., cv. White Sim) flowers advancedsenescence symptoms, including ethylene production and wilting.Pretreatment with amino-oxyacetic acid (AOA) resulted in anincrease of the resistance of the flowers to water stress: waterloss during stress was reduced, recovery was more rapid andwilting was delayed. Water stress accelerated the decrease inlevel of membrane phospholipids, but pretreatment with AOA counteractedthis effect. Since the content of membrane sterols was not affectedby the treatments, the mole ratio of sterol to phospholipidincreased in water-stressed flower petals but not in stressedflowers pretreated with AOA. Membrane permeability and fluiditywere also adversely affected by water stress and AOA: waterstress alone resulted in an increase in permeability and a decreasein fluidity, but in AOA-pretreated stressed flower petals theseparameters were similar to those of nonstressed control flowerpetals. On the basis of these results two main conclusions can be drawn:(a) Water stress induces alterations in the physical and compositionalproperties of carnation petal membranes, (b) Pretreatment ofthe flowers with AOA influences petal membrane traits, mostprobably via modifications in phospholipid turnover, in a waywhich counteracts the effects of water stress. Key words: Amino-oxyacetic acid, Water stress, Carnation flowers     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

Role of Aminocyclopropane-1-carboxylic Acid (ACC) in Control of Ethylene Production in Fresh and Cold-stored Rose Flowers

The relationships between ethylene production, aminocyclopropane-1-carboxylicacid (ACC) content and ethylene-forming-enzyme (EFE) activityduring ageing and cold storage of rose flower petals (Rose hybridaL. cv. Gabriella) were investigated. During flower ageing at20 °C there was a climacteric rise in petal ethylene production,a parallel increase in ACC content, but a continuous decreasein EFE activity. Applied ACC increased petal ethylene productionc. 200-fold. During cold storage of flowers at 1 °C therewere parallel increases in petal ethylene production and ACCcontent, to levels greater than those reached in fresh flowersheld at 20 °C. EFE activity decreased during storage. Immediatelyafter cold-stored flowers were transferred to 20 °C ethyleneproduction and ACC levels were c. four times greater than infreshly cut flowers. These levels increased to maximum valuesof two to four times the maximum values reached during ageingof fresh, unstored, flowers. It was concluded that in rose petalsethylene synthesis is probably regulated by ACC levels and thatcold storage stimulates ethylene synthesis because it increasesthe levels of ACC in the petals. Key words: Rose flower, senescence, ethylene     

The Relationship Between the Change of Calmodulin Content and Ethylene Biosynthesis of Carnation Flower During Its Senescence

The relationship between the change of calmodulin content and the ethylene hiosynthesis in cut carnation flower (Dianthus caryophyllus ‘sun besm’ ) during its senescence was studied. Ethylene releasing was detected at the forth day and reached its peak at the sixth day after the cut carnation flower was cultured under controlled conditions of 27 ℃ with a 14 h photoperiod of 15000 lx provided by fluorescent lamps. The change of calmodulin content positively correlated with the increased' content of ACC, the activity of ACC synthase and ethylene production. The calmodulin contents in petals of the detached flower treated with GA, silver thiosulfate (STS) and aminooxyacetic acid (AOA) were lower than those in control flower petals before they withered, ethylene releasing was reduced and their senescence was delayed too. Ca2+ stimulated ethylene releasing in carnation flower petals, but chlorobenzene (CPZ), an antagonist of calmodulin, inhibited its releasing. It seemed that calmodulin was involved in the regulation of senescence of carnation flower.     

Role of ethylene in the senescence of isolated hibiscus petals

Senescence of petals isolated from flowers of Hibiscus rosa-sinensis L. (cv Pink Versicolor) was associated with increased ethylene production. Exposure to ethylene (10 microliters per liter) accelerated the onset of senescence, as indicated by petal in-rolling, and stimulated ethylene production. Senescence was also hastened by basal application of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminooxyacetic acid, an inhibitor of ethylene biosynthesis, effectively inhibited ethylene production by petals and delayed petal in-rolling. In marked contrast to these results with mature petals, immature petals isolated from flowers the day before flower opening did not respond to ethylene in terms of an increase in ethylene production or petal in-rolling. Furthermore, treatment with silver thiosulfate the day before flower opening effectively prevented petal senescence, while silver thiosulfate treatment on the morning of flower opening was ineffective. Application of ACC to both immature and mature petals greatly stimulated ethylene production indicating the presence of an active ethylene-forming enzyme in both tissues. Immature petals contained less free ACC than mature, presenescent petals and appeared to possess a more active system for converting ACC into its conjugated form. Thus, while the nature of the lack of responsiveness of immature petals to ethylene is unknown, ethylene production in hibiscus petals appears to be regulated by the control over ACC availability.     

Cytokinins in cut carnation flowers. II. Relationship between endogenous ethylene and cytokinin levels in the petals

Tentative identification using HPLC and RIA techniques indicated the presence of zeatin-O-glucoside, zeatin, ribosylzeatin, dihydrozeatin, iso-pentenyladenine and iso-pentenyladenosine in the petals of carnation flowers. Dihydrozeatin is apparently responsible for most of the biological activity. Within the petals most activity was detected in the basal parts which also senesced much slower than the upper parts of the petals. Treatment with AOA extended petal longevity and reduced ethylene production. This was associated with higher cytokinin-like activity in the basal parts of the petals.These higher levels of cytokinins were not observed in the petals of ACC treated flowers or in the detached control flowers. It is suggested that cytokinin transport and/or metabolism may play an important role in regulating ethylene production in cut carnations.     

Involvement of ethylene in the disease caused by Botrytis cinerea on rose and carnation flowers and the possibility of control*

Ethylene production by flowers, petals and leaves of rose was correlated with severity of grey mould. However, when the host became completely macerated, ethylene production diminished. Ethylene production by Botrytis cinerea grown on autoclaved flowers which were supplemented with methionine was negligible. Methionine spray, incubation with ethylene, or precooling of flowers at 4°C increased disease incidence considerably. Ethylene also induced susceptibility of carnation flowers to attack by B. cinerea. On the other hand, sprays of silver thiosulphate (STS) aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) decreased disease severity in rose petals and leaves inoculated with mycelial plugs or conidia. Treatment of cut rose flowers with STS (by dipping) or AOA (by spraying) significantly decreased disease incidence during subsequent incubation at 20 and 10°C. This suggests a treatment for reducing grey mould damage in flowers transported overseas.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes from senescing carnation flowers

Isolated membranes from the petals of senescing carnation flowers (Dianthus caryophyllus L. cv. White-Sim) catalyze the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. A microsomal membrane fraction obtained by centrifugation at 131,000 g for 1 h proved to be more active than the membrane pellet isolated by centrifugation at 10,000 g for 20 min. The ethylene-producing activity of the microsomal membranes is oxygen-dependent, heat-denaturable, sensitive to n-propyl gallate, and saturable with ACC. Corresponding cytosol fractions from the petals are incapable of converting ACC to ethylene. Moreover, the addition of soluble fraction back to the membrane fraction strongly inhibits the ACC to ethylene conversion activity of the membranes. The efficiency with which isolated membranes convert ACC to ethylene is lower than that exhibited by intact flowers based on the relative yield of membranes per flower. This may be due to the presence of the endogenous soluble inhibitor of the reaction, for residual soluble fraction inevitably remains trapped in membrane vesicles isolated from a homogenate.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminoxyacetic acid - AVG aminoethoxyvinylglycine - EPPS N-2-hydroxyethylpiperazine propane sulfonic acid     

The site of 1-aminocyclopropane-1-carboxylic acid synthesis in senescing carnation petals

To study the cause of the uneven production of ethylene by upper and basal portions of detached petals of carnation ( Dianthus caryophyllus L. cv. White Sim), the petals were divided and exposed to ethylene (30 μl 1^-1 for 16 h). The treatment induced rapid wilting and autocatalytic ethylene production in the basal portion similar to that induced in entire petals. In contrast to the response in entire petals and the basal portions, the upper portions responded to ethylene by delayed wilting and much lower ethylene production. Aminocyclopropane carboxylic acid (ACC)-synthase activity in the basal portion of the petals was 38 to 400 times that in the upper portion. In untreated detached petal pieces from senescing carnation flowers, ethylene production by the upper portion declined after 6 h while the basal portion was still producing ethylene at a steady rate 18 h later. Application of ACC to the upper portion of senescing petals increased their ethylene production. α-Aminooxyacetic acid (0.5 m M ), reduced the ethylene production of the senescing basal portion more than that of the upper portion. Endogenous ACC content in basal portions of senescing carnation petals was 3 to 4 times higher than in the upper parts. When detached senescing petals were divided immediately after detaching, the endogenous ACC levels in upper portions remained steady or declined during 24 h after division, while in the basal portions the ACC level rose steadily as in the intact petals. There was no change in the conjugated ACC in either portion after 24 h. Benzyladenine (BA) applied as a pretreatment to entire preclimacteric petals greatly reduced the development of ACC-synthase activity of the basal portion, but had little effect on the activity in the upper portion of the petal. In both portions, however, BA effectively reduced the conversion of ACC to ethylene.     

Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers

2-Aminooxyisobutyric acid (AOIB) has a partial structure of aminooxyacetic acid (AOA) in its whole structure, and resembles 2-aminoisobutyric acid (AIB) in their tetrahedral structures. Both AOA and AIB are inhibitors of ethylene biosynthesis; AOA inhibits the action of 1-aminocyclopropane-1-carboxylate (ACC) synthase and AIB inhibits that of ACC oxidase. The present study showed that AOIB inhibited the in vitro activities of both ACC synthase and ACC oxidase, which were synthesized heterologously in E. coli cells from corresponding carnation cDNAs, and the magnitudes of inhibition were similar to those caused by AOA and AIB; AOIB and AOA at 0.1 mM inhibited ACC synthase action by 75%, and AOIB and AIB at 10 mM inhibited ACC oxidase action by 16.3 and 22.5%, respectively. AOIB at 1 mM caused 91.5% reduction of maximum ethylene production rate as compared to the control in cut ‘Excerea’ carnation flowers undergoing senescence, thereby lengthening their vase life to 7 d from 3 d of the control flowers. The inhibition by AOIB was probably caused by its action resembling AOA, but not AIB. AOIB also extended significantly the vase life of cut flowers of ‘Pax’ carnation, and tended to do so in ‘Primero Mango’ carnation. The present findings suggest the potential of AOIB as a new preservative for carnations and other ornamentals in which ethylene plays a key role in the induction of senescence.     

1-Aminocyclopropane-1-Carboxylic Acid Transported from Roots to Shoots Promotes Leaf Abscission in Cleopatra Mandarin (Citrus reshni Hort. ex Tan.) Seedlings Rehydrated after Water Stress

The effect of water stress and subsequent rehydration on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity, ethylene production, and leaf abscission was studied in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings. Leaf abscission occurred when drought-stressed plants were allowed to rehydrate, whereas no abscission was observed in plants under water stress conditions. In roots of water-stressed plants, a high ACC accumulation and an increase in ACC synthase activity were observed. Neither increase in ACC content nor significant ethylene production were detected in leaves of water-stressed plants. After rehydration, a sharp rise in ACC content and ethylene production was observed in leaves of water-stressed plants. Content of ACC in xylem fluid was 10-fold higher in plants rehydrated for 2 h after water stress than in nonstressed plants. Leaf abscission induced by rehydration after drought stress was inhibited when roots or shoots were treated before water stress with aminooxyacetic acid (AOA, inhibitor of ACC synthase) or cobalt ion (inhibitor of ethylene-forming enzyme), respectively. However, AOA treatments to shoots did not suppress leaf abscission. The data indicate that water stress promotes ACC synthesis in roots of Cleopatra mandarin seedlings. Rehydration of plants results in ACC transport to the shoots, where it is oxidized to ethylene. Subsequently, this ethylene induces leaf abscission.     

Physiological Responses of Cut Rose Flowers to Exposure to Low Temperature: Changes in Membrane Permeability and Ethylene Production

Senescence of cut rose flowers (Rosa hybrida L. cv. Mercedes)at 22 °C occurred earlier in flowers previously held at2 °C for 10 d or 17 d than in freshly cut flowers. Thisadvanced senescence was observed as an earlier increase in bothethylene production rate and membrane permeability. The risein ethylene production preceded the rise in the level of ionleakage from petals, and this in turn preceded visible symptomsof petal death. Applied ethylene stimulated ion leakage andinhibitors of ethylene synthesis and action (amino-oxyaceticacid and silver thiosulphate respectively) inhibited the normalincrease in ion leakage. The maximum rate of ethylene productionof 22 °C increased markedly in petals of flowers previouslyheld at 2 °C, up to nine times the level in fresh flowers.We conclude that during exposure of rose flowers to 2 °C,in addition to senescence, processes were induced which ledto stimulated ethylene production after transferral to 22 °C.Ethylene apparently caused the subsequent advance in membranepermeability and senescence. Key words: Rose flower, Low temperature, Senescence     

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.     

The mechanism involved in ethylene-enhanced ethylene synthesis in carnations

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

The Role of Ethylene in Interorgan Signaling during Flower Senescence

The roles of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene in interorgan signaling during senescence in orchid (Cymbidium) flowers were investigated. Following application of radiolabeled ACC to the stigma or the rostellum (modified lobe of the stigma), radiolabeled ethylene is produced by all flower parts. In intact flowers as well as in excised central columns, stigma- or rostellum-applied ACC or [alpha]-aminoisobutyric acid were largely immobile. Local treatment of the central column of previously aminoethoxyvinylglycine-treated flowers with either ethylene or 2-chloroethylphosphonic acid (ethephon) rapidly induced emission of ethylene from the petals, showing that ethylene is readily translocated within the flower. Creation of alternative outlets (incisions) in the labellum or the central column significantly delayed the occurrence of senescence symptoms in ACC-treated flowers. The results do not confirm the presumed role of ACC as a signal in interorgan communication during flower senescence. In these flowers, ethylene produced in the stigmatic region following pollination or emasculation serves as a mobile factor responsible for senescence symptoms observed in other flower parts.     

Inhibition by 1-Aminocyclobutane-l-Carboxylate of the Activity of 1-Aminocyclopropane-l-Carboxylate Oxidase Obtained from Senescing Petals of Carnation {Dianthus caryophyllus L.) Flowers

We partially purified 1-aminocyclopropane-l-carboxy-late (ACC)oxidase from senescing petals of carnation {Dianthus caryophyllusL. cv. Nora) flowers and investigated its general characteristics,and, in particular, the inhibition of its activity by ACC analogs.The enzyme had an optimum pH at 7-7.5 and required Fe²⁺, ascorbateand NaHCO₃ for its maximal activity. The K_m for ACC was calculatedas 111-125 µM in the presence of NaHCO₃. Its M_r was estimatedto be 35 and 36 kDa by gel-filtration chromatography on HPLCand SDS-PAGE, respectively, indicating that the enzyme existsin a monomeric form. These properties were in agreement withthose reported previously with ACC oxidases from different planttissues including senescing carnation petals. Among six ACCanalogs tested, l-aminocyclobutane-l-carboxylate (ACBC) inhibitedmost severely the activity of ACC oxidase from carnation petals.ACBC acted as a competitive inhibitor with the K_i of 20-31 µM.The comparison between the K_m for ACC and the K_i for ACBC indicatedthat ACBC had an affinity which was ca. 5-fold higher than thatof ACC. Whereas ACC inactivated carnation ACC oxidase in a time-dependentmanner during incubation, ACBC did not cause the inactiva-tionof the enzyme. Preliminary experiments showed that ACBC andits N-substituted derivatives delayed the onset of senescencein cut carnation flowers. (Received August 19, 1996; Accepted November 26, 1996)     

Interrelationship between the Different Flower Parts during Emasculation-Induced Senescence in Cymbidium Flowers

In Cymbidium flowers emasculation by removal of the anther capand the pollinia, led to rapid colouration of the lip and advancedwilting of the petals and sepals. The ethylene production ofwhole flowers showed an emasculation-induced early peak in ethyleneevolution followed some days later by a second increase concomitantwith the wilting of the flower. In non-emasculated flowers theethylene production increased later and simultaneously withcolouration of the lip and wilting of the petals and sepals.At all stages of senescence, the contribution of the lip, petals,and sepals to the total amount of ethylene produced was negligible. Parallel to the increase in ethylene production of whole flowers,an increase in 1-aminocyclopropane-l-carboxylic acid (ACC) andmalonyl-ACC (MACC) in the central column and, to a lesser extent,in the ovary was observed. Also an increase in internal ethyleneconcentration was demonstrated and this, in contrast, was apparentin all the different flower parts. The activity of the ethylene-formingenzyme in lips, petals, and sepals showed an increase afteremasculation and such an effect could also be induced by treatmentof isolated lips with low concentrations of ethylene. The data indicate that senescence in Cymbidium flowers is regulatedby the central column and perhaps the ovary and that both ACCand ethylene may play a signalling role in inter-organ communication. Key words: 1-aminocyclopropane-l-carboxylic acid, ethylene, Cymbidium, senescence     

Intracellular energy depletion triggers programmed cell death during petal senescence in tulip

Programmed cell death (PCD) in petals provides a model system to study the molecular aspects of organ senescence. In this study, the very early triggering signal for PCD during the senescence process from young green buds to 14-d-old petals of Tulipa gesneriana was determined. The opening and closing movement of petals of intact plants increased for the first 3 d and then gradually decreased. DNA degradation and cytochrome c (Cyt c) release were clearly observed in 6-d-old flowers. Oxidative stress or ethylene production can be excluded as the early signal for petal PCD. In contrast, ATP was dramatically depleted after the first day of flower opening. Sucrose supplementation to cut flowers maintained their ATP levels and the movement ability for a longer time than in those kept in water. The onset of DNA degradation, Cyt c release, and petal senescence was also delayed by sucrose supplementation to cut flowers. These results suggest that intracellular energy depletion, rather than oxidative stress or ethylene production, may be the very early signal to trigger PCD in tulip petals.