Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs. Received: 18 June 1998 / Accepted: 22 December 1998     

Does Pollination Induce Corolla Abscission of Cyclamen Flowers by Promoting Ethylene Production?

Very low ethylene production rates were measured in nonpollinated Cyclamen persicum Mill flowers, and no change in production was observed during the whole life span of the flower until death. Normal senescence was accompanied by a gradual discoloration and loss of turgor followed by wilting. Pollination induced a dramatic increase in ethylene evolution, culminating in a peak 4 days after pollination, and abscission of the corolla on that day. Silver-thiosulfate, an inhibitor of ethylene action, had no effect on longevity of unpollinated flowers, but completely nullified the effect of pollination on corolla abscission. Exposing unpollinated flowers to very high ethylene concentrations (50 microliters per liter) for 48 hours did not promote corolla abscission or senescence. 1-Aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene, increased ethylene production by unpollinated flowers more than 100-fold, but did not promote corolla abscission. 1-Aminocyclopropane-1-carboxylic acid did enhance corolla abscission of pollinated flowers. It is concluded that the main effect of pollination in inducing corolla abscission of cyclamen is by rendering the tissue sensitive to ethylene, apart from the promotion of ethylene production.     

Sites of ethylene production in the pollinated and unpollinated senescing carnation (Dianthus caryophyllus) inflorescence

Production of endogenous ethylene from the styles, ovary and petals of pollinated and unpollinated flowers of Dianthus caryophyllus L. was measured. The rate of ethylene production of cut, unpollinated flowers aged in water at 18°C was low until the onset of petal wilting, when a rapid surge of ethylene occurred in all tissues. The flower ethylene production was evolved mostly from the styles and petals. The bases of petals from unpollinated, senescing flowers evolved ethylene faster and sometimes earlier than the upper parts. Treatment of cut flowers with propylene, an ethylene analogue, accelerated wilting of flower petals and promoted endogenous ethylene production in all flower tissues. Pollination of intact flowers also promoted endogenous ethylene production and caused accelerated petal wilting within 2–3 days from pollination. Although the data are consistent with the hypothesis that ethylene forms a link between pollination of the style and petal wilting, in the unpollinated flower the style and petals can evolve a surge of ethylene independently of each other, about the time when the petals irreversibly wilt. The results are discussed in relation to the role of ethylene in flower 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 effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

Post-pollination changes in the floral organs of two <Emphasis Type="Italic">Cymbidium</Emphasis> species

It was observed that the unpollinated flowers of Cymbidium pendulum (Roxb.) Sw. and C. aloifolium (L.) Sw. stayed fresh for 20 and 18 d, respectively, but attained senescence in 8 and 7 d, respectively, after pollination. The higher content of total soluble sugars, reducing sugars and free amino acids was observed in all the floral organs of pollinated flowers than in unpollinated ones. Pollination also up-regulated the activity of hydrolytic (α-amylase, β-amylase, invertase) and proteolytic enzymes (proteases) in floral organs. Amongst floral organs, the lip and perianth possessed highest contents of metabolites. Application of auxin inhibitor (0.25 μM triiodobenzoic acid) and ethylene inhibitor (0.25 μM AgNO₃) to the pollinated flowers partially prevented the process of senescence.     

Lack of Control by Early Pistillate Ethylene of the Accelerated Wilting of Petunia hybrida Flowers

Well before pollen tube penetration, ethylene has begun to disseminate from pollinated styles of Petunia hybrida flowers. Previous stigmatic application of aminoethoxyvinylglycine (AVG) completely prevented this ethylene synthesis, indicating that the endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen is not readily converted on the stigma. Compared to other flower parts, the capacity of the ethylene forming enzyme was largest in the stigma. When applied to the stigma, ACC caused ethylene synthesis, but did not accelerate wilting, unless high concentrations (20 nanomols) were used. Upon pollination or stigma wounding, the early ethylene evolved exclusively from the gynoecium, much later followed by the synthesis of corolla ethylene. Employing wideneck Erlenmeyer flasks, the competitive inhibitor of ethylene action, norbornadiene, was applied to entire flowers in situ, with delaying effects on wound-induced wilting. In contrast, norbornadiene treatment of styles alone, using capillaries, could not postpone wilting. Pollination with foreign pollen species did not lead to accelerated corolla wilting, notwithstanding considerable synthesis of ethylene during the first 5 hours. In situ treatment of the stigma with AVG considerably delayed wound- and pollination-induced wilting. Removal of the entire AVG-treated style 6 hours after stigma wounding still allowed for the postponement of the accelerated wilting, even at very low concentrations of AVG. It is concluded that early stylar ethylene does not play a role in the acceleration of wilting but that, much later, corolla ethylene does, induced by a mobile wilting factor from the stigma, which is ACC.     

Studies on flower longevity in Digitalis

The flowers of Digitalis purpurea respond to pollination by rapid corolla abscission without any loss of corolla turgor, nor any significant loss of corolla constituents, relative to the corollas of unpollinated flowers of a similar age. The corollas of unpollinated flowers too eventually abscise, 6 d after the stigma opens, however, they do so with only a minimal loss of fresh weight or corolla constituents. Pollination causes an increase in ethylene production detectable within 1 h. Increased ethylene production occurs initially only from the upper portion of the style, later from the lower portion, and lastly, between 23 and 48 h after pollination, from the ovary plus calyx. The pollination response can be induced by exogenous ethylene, the degree of weakening of the corolla abscission zone being dependent upon the concentration and duration of the exposure period and on the stage of flower development. The regulation of ethylene biosynthesis and its involvement in the control of pollination-induced corolla abscission are discussed.     

Ethylene sensitivity: The role of short-chain saturated fatty acids in pollination-induced senescence of Petunia hybrida flowers

Normal and pollination-induced senescence of Petunia hybrida L cv. Pink Cascade flowers is accompanied by an increase in the sensitivity of the corolla to ethylene as indicated by an acceleration in the rate of corolla bluing after exposure to exogenous ethylene. Pollination resulted in the production of short-chain saturated fatty acids ranging in chain length from C₆ to C₁₀. Following pollination, these acids are synthesized in the stylar tissue via the acetate pathway within the first 12 hours. The fatty acids are transported rapidly to the corolla where they induce an increase in ethylene sensitivity. In unpollinated flowers, these acids are produced in the corolla during the early stages of senescence. Although the levels of these fatty acids decrease rapidly during the final stages of senescence, a significant increase in ethylene sensitivity could be detected prior to the decrease. It appears that the increase in ethylene sensitivity caused by the synthesis of short-chain saturated fatty acids occurs concurrently, but independent from ethylene synthesis.     

Isolation of an 1-aminocyclopropane-1-carboxylate oxidase gene from mulberry (Morus alba L.) and analysis of the function of this gene in plant development and stresses response

Mulberry (Morus alba) is an important crop tree involved in sericulture and pharmaceuticals. To further understand the development and the environmental adaptability mechanism of mulberry, a cDNA of the gene MaACO1 encoding 1-aminocyclopropane-1-carboxylate oxidase was isolated from mulberry. This was used to investigate stress-responsive expression in mulberry. Developmental expression of ACC oxidase in mulberry leaves and spatial expression in mulberry flowers were also investigated. Damage and low-temperature treatment promoted the expression of MaACO1 in mulberry. In leaves, expression of the MaACO1 gene increased in cotyledons and the lowest leaves with leaf development, but showed reduced levels in emerging leaves. In flowers, the pollinated stigma showed the highest expression level, followed by the unpollinated stigma, ovary, and immature flowers. These results suggest that high MaACO1 expression may be predominantly associated with tissue aging or senescence in mulberry.     

Pollination and stigma wounding: same response, different signal?

In Petunia hybrida flowers, both pollination and stigma woundinginduced a transient Increase in ethylene production and hastenedcorolla senescence. Ethylene production by different flowerparts was measured in situ using laser photoacoustic (LPA) spectroscopy.In pollinated flowers, ethylene was exclusively produced bythe stigma/style region whereas wounding of the stigma Inducedethylene production both by the stigma/style region and by theremaining flower parts. In aminoethoxyvinylglycine (AVG)-treatedflowers, subsequent treatment of the unwounded stigma with 1-aminocyclopropane-1-carboxylicacid (ACC) induced ethylene production exclusively by the stigma/styleregion whereas treatment of a previously wounded stigma withACC induced a simultaneous increase in ethylene production bythe stigma/style region and the remaining flower parts. Theseresults suggest that following stigma wounding, either ACC orethylene is involved in inter-organ communication. Followingpollination, the signal is apparently not directly related toethylene. In vivo ACC oxidase activity of most flower parts, includingthe gynoecium, was higher in light than in dark. Light or darkdid not influence the relative contributions of stigma/styleand remaining flower parts to the total pollination, woundingor ACC-induced ethylene production, indicating that ACC is nottranslocated. Both in excised styles and intact flowers, radiolabelledACC and its analogue -aminoisobutyric acid (AIB), applied eitherto an intact or wounded stigma, were largely immobile confirmingthat ACC is not likely to play a role in inter-organ signalling. The results collectively suggest that following stigma wounding,translocation of ethylene may be the signal responsible forinitiation of corolla senescence; following pollination thesignal is not directly related to ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid (ACC), ethylene, flower senescence, Petunia hybrida, pollination, stigma wounding     

Pistil-Specific and Ethylene-Regulated Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes in Petunia Flowers

The differential expression of the petunia 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family during flower development and senescence was investigated. ACC oxidase catalyzes the conversion of ACC to ethylene. The increase in ethylene production by petunia corollas during senescence was preceded by increased ACC oxidase mRNA and enzyme activity. Treatment of flowers with ethylene led to an increase in ethylene production, ACC oxidase mRNA, and ACC oxidase activity in corollas. In contrast, leaves did not exhibit increased ethylene production or ACC oxidase expression in response to ethylene. Gene-specific probes revealed that the ACO1 gene was expressed specifically in senescing corollas and in other floral organs following exposure to ethylene. The ACO3 and ACO4 genes were specifically expressed in developing pistil tissue. In situ hybridization experiments revealed that ACC oxidase mRNAs were specifically localized to the secretory cells of the stigma and the connective tissue of the receptacle, including the nectaries. Treatment of flower buds with ethylene led to patterns of ACC oxidase gene expression spatially distinct from the patterns observed during development. The timing and tissue specificity of ACC oxidase expression during pistil development were paralleled by physiological processes associated with reproduction, including nectar secretion, accumulation of stigmatic exudate, and development of the self-incompatible response.     

Post-pollination biochemical changes in the floral organs of<Emphasis Type="Italic">Rhynchostylis retusa</Emphasis> (L.) Bl. and<Emphasis Type="Italic">Aerides multiflora</Emphasis> Roxb. (Orchidaceae)

If left unpollinated, the flowers ofAerides multiflora (Roxb.) andRhynchostylis retusa (L.) Bl. can remain fresh for 17 and 24 d, respectively. However, they begin to wilt at 2 to 3 days after pollination (DAP) and 3 to 4 DAP, respectively, and become senescent at 5 DAP and 7 DAP, respectively. When measured at two developmental phases — Stage 1, start of wilting and Stage 2, progression to senescence — all the floral organs from pollinated flowers had higher contents of total soluble sugars, reducing sugars, and free amino acids than those from unpollinated flowers. A corresponding increase was noted in the activities of hydrolytic enzymes, i.e., α-amylase, β-amylase, and invertase, and proteolytic enzymes (proteases) in those organs. This indicated that signals related to pollination had up-regulated those activities, leading to a breakdown of complex molecules into simpler ones for mobilization. The amounts of sugars and enzyme activity were relatively greater in the pollinated flowers ofA. multiflora compared withR. retusa, and levels were always higher in the floral lips and perianths. When inhibitors of auxin (0.25 mM TIBA) or ethylene (0.25 mM AgNO₂) were applied to the pollinated flowers, their senescence was partially prevented, thus signifying hormonal involvement in governing the pollination-induced biochemical alterations normally found in those organs.     

Ethylene Synthesis and Floral Senescence following Compatible and Incompatible Pollinations in Petunia inflata

Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata. Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hours after pollination. P. inflata pollen was found to carry large amounts of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The amount of pollen-held ACC varied in different genetic backgrounds, and the magnitude of the peak correlated with the amount of ACC borne by the pollen. Aminooxyacetic acid (AOA), an inhibitor of ACC synthesis, had no inhibitory effect on this ethylene response, indicating that pollen-borne ACC was largely responsible for the early synthesis of ethylene. After compatible pollination, a second increase in ethylene synthesis began at 18 hours, and the first sign of senescence appeared at 36 hours. Upon treatment with AOA, the second phase of ethylene production was reduced by 95%, indicating that endogenous ACC synthesis was required for this phase of ethylene synthesis. AOA treatment also delayed senescence to 6 days after anthesis. After incompatible pollination, a second increase in ethylene production did not occur until 3 days, and the first sign of senescence occurred 12 hours later. Unpollinated flowers showed an increase in ethylene production 3 to 4 days after anthesis and displayed signs of senescence 1 day later. The significance of the early and late phases of pollination-induced ethylene synthesis is discussed.     

Temporal and spatial regulation of 1-aminocyclopropane-1-carboxylate oxidase in the pollination-induced senescence of orchid flowers.

Pollination of many flowers initiates a sequence of precisely regulated developmental events that include senescence of the perianth and development of the ovary. The plant hormone ethylene is known to play a key role in regulating the biochemical and anatomical changes that constitute the postpollination syndrome. For this reason, we have studied the pollination syndrome in Phalaenopsis orchids by examining the spatial and temporal location of ethylene biosynthesis within the orchid flower, and how this biosynthesis is regulated by factors that influence expression of genes that encode key enzymes in the ethylene biosynthetic pathway. In particular, we examined the role in the postpollination syndrome of the expression of the gene for 1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyzes the conversion of ACC to ethylene. In vivo incubation of tissues with the ethylene precursor ACC demonstrated that ACC oxidase activity increases after pollination in the stigma, contrary to the observation that activity is constitutive in petunia and carnation gynoecia. RNA blot hybridization of floral tissues indicates that the increase in ACC oxidase activity is due to de novo synthesis of mRNA and presumably protein, which is induced after pollination. Furthermore, the pattern of induction is consistent with a model of coordinate regulation of gene expression in which the pollination signal travels to other organs of the flower to induce their ethylene production. We have also used in situ hybridization to define further the temporal and spatial expression of ACC oxidase within the floral organs, showing that expression, and,by inference, the capability to oxidize ACC to ethylene, is induced in all living cells of the tissues examined after pollination. These findings contrast with work in petunia that suggests that ACC oxidase is localized to the stigmatic surface.