Effects of various chemical agents and early ethylene production on floral senescence of Hibiscus syriacus L.

To understand the factors that induce floral senescence in Hibiscus syriacus L., we have investigated the effects of various chemical agents on flower senescence at two different flowering stages, before and after full bloom, as well as the relationship between flower longevity and endogenous ethylene production before full bloom. Treatments with ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ethephon enhanced floral senescence, while aminoethoxyvinylglycine (AVG) promoted flower longevity regardless of treatment timing. Although ethanol slightly extended flower longevity, abscisic acid (ABA), nitric oxide, boric acid and sucrose, which have been reported to affect flower longevity or senescence, had no effect on H. syriacus floral senescence. The polyamine spermine (SPM), methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SPM biosynthesis, and cycloheximide (CHI) accelerated flower senescence when applied before full bloom, but had no effect when applied after full bloom. SPM, MGBG and CHI treatments resulted in enhanced ethylene production during flower opening, and the promotion of flower senescence is mediated by ethylene production prior to full bloom. Furthermore, endogenous ethylene, spontaneously produced before blooming, was closely associated with floral senescence. These results suggest that ethylene production during flower opening plays a key role in determining the timing of Hibiscus flower senescence.     

Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

Effects of Promoters and Inhibitors of Ethylene and ABA on Flower Senescence of Hibiscus rosa-sinensis L.

Hibiscus rosa-sinensis L. flowers (cv La France) senesce and die over a 12-h period after opening. The aim of this study was to examine the physiological mechanisms regulating the senescence process of ephemeral hibiscus flowers. Different flower stages and floral organs were used to determine whether any interaction existed during flower senescence between endogenous abscisic acid (ABA) and the predisposition of the tissue to ethylene synthesis. This was carried out on whole flowers treated with promoters and inhibitors of ethylene and ABA synthesis or a combination of them. Treatments with 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene biosynthesis, enhanced flower senescence, whereas amino-oxyacetic acid (AOA) and fluridone, an ethylene and an ABA inhibitor, respectively, extended flower longevity. These effects were more significant when applied before anthesis. Ethylene evolution was substantially reduced in all organs from open and senescent flowers treated with fluridone and AOA. Similarly, endogenous ABA accumulation was negatively affected by AOA and fluridone treatments. Application of fluridone plus ACC reduced ethylene evolution and increased ABA content in a tissue-specific manner but did not overcome the inhibitor effect on flower longevity. AOA plus fluridone treatment slightly accelerated flower longevity compared to AOA-treated flowers. Application of ABA alone promoted senescence, suppressed ethylene production, and, when applied with fluridone, countered the fluridone-induced increase in flower longevity. Taken together, these results suggest that the senescence of hibiscus flowers is an endogenously regulated ethylene- and ABA-dependent process.     

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     

Inhibition of wilting and autocatalytic ethylene production in cut carnation flowers by cis-propenylphosphonic acid

The effect of cis-propenylphosphonic acid (PPOH), a structural analoge of ethylene, on flower wilting and ethylene production was investigated using cut carnation flowers which are very sensitive to ethylene. Wilting (petal in-rolling) of the flowers was delayed by continuously immersing the stems in a 5–20 mM PPOH solution. In addition, the continuous treatment with PPOH markedly reduced autocatalytic ethylene production of the petals accompanying senescence. This reduction of autocatalytic ethylene production was considered responsible for the inhibitory effect of PPOH on flower wilting. The inhibitory activity of trans-propenylphosphonic acid (trans-PPOH), on both flower wilting and the autocatalytic ethylene production accompanying senescence was markedly lower than that of PPOH, suggesting that PPOH action is stereoselective. PPOH may be of interest as a new, water-soluble inhibitor of wilting and autocatalytic ethylene production in cut carnation flowers.     

Spatial and temporal distribution of mineral nutrients and sugars throughout the lifespan of <Emphasis Type="Italic">Hibiscus rosa-sinensis</Emphasis> L. flower

Although the physiological and molecular mechanisms of flower development and senescence have been extensively investigated, a whole-flower partitioning study of mineral concentrations has not been carried out. In this work, the distribution of sucrose, total reducing sugars, dry and fresh weight and macro and micronutrients were analysed in Hibiscus rosa-sinensis L. petals, stylestigma including stamens and ovary at different developmental stages (bud, open and senescent flowers). Total reducing sugars showed the highest value in petals of bud flowers, then fell during the later stages of flower development whereas sucrose showed the highest value in petals of senescent flowers. In petals, nitrogen and phosphorus content increased during flower opening, then nitrogen level decreased in senescent flowers. The calcium, phosphorus and boron concentrations were highest in ovary tissues whatever the developmental stage. Overall, the data presented suggests that the high level of total reducing sugars prior the onset of flower opening contributes to support petal cells expansion, while the high amount of sucrose at the time of petal wilting may be viewed as a result of senescence. Furthermore, this study discusses how the accumulation of particular mineral nutrients can be considered in a tissue specific manner for the activation of processes directly connected with reproduction.     

Physiological changes accompanying senescence in the ephemeral daylily flower

The daylily flower, Hemerocallis hybrid cv Cradle Song, develops from the opening bud to full senescence in 36 hours. Unlike other ephemeral flowers studied to date, it does not respond to ethylene, but other senescence phenomena are similar. There was a small respiration climacteric coinciding with early flower senescence, and it was also observed in isolated petals and petal slices. Cycloheximide abolished the climacteric and delayed senescence in all three systems. Petal apparent free space increased from 30% at bud opening to 38% at the onset of senescence, and sugar efflux increased from 0.2 to 2.8 milligrams per gram of fresh weight per hour during the same period. A sharp increase in ion efflux from 0.8 to 4.0 micromoles of NaCl equivalents per gram of fresh weight per hour, coinciding with the climacteric, was abolished by cycloheximide. Uptake of radiolabeled inorganic phosphate by petal slices from 100 micromolar solution increased during onset of senescence from 6 to 10 nmoles per gram of fresh weight per hour. Half was esterified; of this, 14% went into ATP, and the cellular energy charge remained high at 0.86 during senescence. The proportion incorporated into phospholipid (2.2%) did not change during senescence, but the proportion in phosphatidyl choline increased and in phosphatidyl glycerol decreased during senescence. The general phosphate ester pattern in presenescent slices closely resembled that in other plant tissues except that phospholipid precursors were more prominent (approximately 20% of total organic ³²P versus 5%). In senescent slices, the proportion of hexose phosphates decreased from 40 to 15% of total organic ³²P and that of phospholipid precursors increased to approximately 50%, suggesting that phospholipid synthesis was blocked early in senescence.     

Increased oxidative stress,lipid peroxidation and protein degradation trigger senescence in <Emphasis Type="Italic">Iris versicolor</Emphasis> L. flowers

Dynamics in various physiological and biochemical aspects were studied during various stages (I—tight bud stage to VI—senescent stage) of flower development in Iris versicolor. Floral diameter, fresh & dry mass and water content increased during flower opening and decreased towards senescence. Senescence was found to be related to the increased lipid peroxidation which was reflected in the decreased membrane stability index towards senescence. This increase in the lipid peroxidation was probably initiated by increased lipoxygenase activity which shot up just prior to the increase in lipid peroxidation. Soluble protein content showed a marginal decrease towards senescence with a corresponding increase in specific protease activity. Sugar fractions and α-amino acids showed a significant decrease towards senescence. Superoxide dismutase and ascorbate peroxidase activity increased as the flowers opened and thereafter a significant decrease was registered towards senescence. Catalase activity improved as the flower matures, but decreased prior to flower opening through senescence. The protein patterns from the tepal tissues resolved through electrophoresis showed a consistency in proteins upto the flower opening but a marginal decrease was registered in both high and low molecular weight proteins towards senescence. However, a protein of molecular weight 76.5 kDa showed up during senescent stages which may have a role in flower senescence.     

Characterization of proteolytic activity during senescence in daylilies

From 12 to 24 h after the opening of daylily flowers ( Hemerocallis hybrid cv. Stella d'Oro), the petals begin to degrade and the protein levels of soluble, microsomal‐ and plastid‐enriched fractions decrease by 50%, on a per petal basis. To help determine some of the components for the cell death program in daylily petals, we studied the mechanisms that regulate this loss of protein. Enzyme activities capable of digesting native daylily protein, gelatin, and azocasein markedly increase after flower opening, and their appearance is inhibited by the translation inhibitor, cycloheximide. Protein hydrolysis in vitro is prevented by inhibitors of cysteine, serine and metalloproteinases. Immunoblots using antibodies to ubiquitin pathway enzymes indicate that the ubiquitin system is not senescence specific. However, ion leakage is delayed by two inhibitors of the 26S proteasome. We propose that programmed cell death in daylily petals may involve the increase in activity of at least three classes of proteinases, and discuss the possibility that these proteinases may operate in concert with the ubiquitin pathway.     

Galactose metabolism in cell walls of opening and senescing petunia petals

Galactose was the major non-cellulosic neutral sugar present in the cell walls of ‘Mitchell’ petunia (Petunia axillaris × P. axillaris × P. hybrida) flower petals. Over the 24 h period associated with flower opening, there was a doubling of the galactose content of polymers strongly associated with cellulose and insoluble in strong alkali (‘residual’ fraction). By two days after flower opening, the galactose content of both the residual fraction and a Na₂CO₃-soluble pectin-rich cell wall fraction had sharply decreased, and continued to decline as flowers began to wilt. In contrast, amounts of other neutral sugars showed little change over this time, and depolymerisation of pectins and hemicelluloses was barely detectable throughout petal development. Size exclusion chromatography of Na₂CO₃-soluble pectins showed that there was a loss of neutral sugar relative to uronic acid content, consistent with a substantial loss of galactose from rhamnogalacturonan-I-type pectin. β-Galactosidase activity (EC 3.2.1.23) increased at bud opening, and remained high through to petal senescence. Two cDNAs encoding β-galactosidase were isolated from a mixed stage petal library. Both deduced proteins are β-galactosidases of Glycosyl Hydrolase Family 35, possessing lectin-like sugar-binding domains at their carboxyl terminus. PhBGAL1 was expressed at relatively high levels only during flower opening, while PhBGAL2 mRNA accumulation occurred at lower levels in mature and senescent petals. The data suggest that metabolism of cell wall-associated polymeric galactose is the major feature of both the opening and senescence of ‘Mitchell’ petunia flower petals.     

Ethylene production by young Aranda orchid flowers and buds

A high rate of ethylene production was observed in buds and young flowers of Aranda orchid, which increased with bud growth, reaching a high value in half-opened flower. This was followed by a gradual decline but it increased again when the flowers showed sign of senescence. Aminooxylacetic acid (AOA) inhibited ethylene production and bud expansion of Aranda buds.     

Physiological Responses of Chrysanthemum Petals during Senescence

Flower senescence was studied in three cultivars of Chrysanthemum coronarium L.: Snowball White, Yellow Chandrama (standard type flowers) and Spray Button (spray type flowers). Spray button flowers exhibited least respiration rate, less efflux of ions, minimum protease activity and less decline in fresh mass, relative water content and total soluble protein content with the progression of senescence. The Snowball White flowers showed highest respiration rate, great efflux of ions, maximum protease activity, high activity of hydrolytic as well as proteolytic enzymes, and high decline in fresh mass, relative water content, and total soluble protein content. Yellow Chandrama flowers showed responses similar to Snowball White flowers.     

Active anthocyanin degradation in <Emphasis Type="Italic">Brunfelsia calycina</Emphasis> (yesterday–today–tomorrow) flowers

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.     

Invertase inhibitor-control of sucrose transport from carnation petals to other flower parts

An investigation was made of the distribution of metabolites in the receptacles and ovaries, and the activity of an invertase inhibitor after feeding the petals with ¹⁴C-sucrose and cycloheximide during the natural aging of cut carnation flower. The cycloheximide treatment blocked the synthesis of the invertase inhibitor in petals and maintained a relatively high level of invertase activity during flower senscence. Treatment with cycloheximide decreased the movement of radioactivity from petals to receptacles and ovaries from 7 to 10 times, compared to non-treated control flowers. IAA applied on the stigma had no influence on synthesis of the inhibitor and distribution of the radioactivity.     

The role of protein synthesis in the senescence of leaves: I. The formation of protease

The senescence of oat leaves has been studied by following the loss of chlorophyll and protein and the increase of α-amino nitrogen, after detachment and darkening. Protein synthesis and the amounts of proteolytic enzymes in the leaves have been determined directly. The process of senescence is shown to be a sequential one in which protein synthesis,most probably the formation of a proteolytic enzyme with l-serine in its active center, is of prime importance. The evidence is as follows. Firstly, l-serine specifically enhances senescence, especially in presence of kinetin. Secondly, cycloheximide, which inhibits protein synthesis in other systems, delays senescence and prevents the serine enhancement. Although requiring higher concentrations, cycloheximide can be as effective as kinetin in inhibiting senescence. It is shown directly that cycloheximide prevents protein synthesis in oat leaves under the same conditions as when it prevents senescence. Thirdly, leaves have been shown to contain two proteinases, with pH optima at 3 and 7.5, whose activity increases during senescence, even though the total leaf protein is decreasing. The amounts of both these enzymes present after 3 days are clearly increased by serine, and are greatly decreased by cycloheximide or by kinetin. The role of kinetin in delaying senescence thus may rest on its ability to suppress protease formation.     

Effects of ethylene and 1-MCP (1-methylcyclopropene) on bud and flower drop in mini <Emphasis Type="Italic">Phalaenopsis</Emphasis> cultivars

Phalaenopsis frequently exhibits bud drop during production and in response to adverse postharvest conditions. The effect of exogenous ethylene on bud drop of mini Phalaenopsis was studied and ethylene sensitivity of four cultivars was compared. Water content, membrane permeability and ABA (abscisic acid) content in floral buds and flowers were determined after ethylene treatment. Exogenous ethylene induced flower bud drop in all tested Phalaenopsis cultivars and the different cultivars showed distinct differences in ethylene sensitivity. The cultivar Sogo ‘Vivien’ exhibited the highest bud drop, water loss and change in membrane permeability in floral petals, while Sogo ‘Berry’ showed the lowest sensitivity. The ethylene inhibitor 1-MCP (1-methylcyclopropene) reduced ethylene-induced floral bud drop in the cultivar Sogo ‘Yenlin’. ABA content in floral buds was increased in response to ethylene and 1-MCP pretreatment inhibited the ethylene-induced increase in ABA levels efficiently. This finding suggests that the observed increase in ABA content during bud drop was mediated by ethylene. The interaction between ABA and ethylene is discussed.     

Flower opening and closing ofOxalis martiana

Flowers ofOxalis martiana open in the morning and close in the afternoon repeatedly for 3–5 days in May–July under natural conditions. Both in light and in darkness, the closed flowers opened in reponse to a rise in temperature (thermonasty), but not under the constant temperatures. Transfer from darkness to light along with temperature rise caused rapid flower opening, and at 20 C or higher temperatures, exposure to light caused flower opening even without changing temperature (photonasty). Therefore, the temperature of the night before opening is critical in determining whether the flower opening under natural conditions depends on thermonasty or photonasty. The opened flowers closed about 8–11 hr after the beginning of opening both under natural conditions and constant light-temperature conditions, which suggests that the time of flower closing is determined by endogenous factors. Length of the perianth increased greatly during opening and slightly during closing. Application of actionomycin D or cycloheximide inhibited both the flower opening and closing, probably by suppressing the perianth growth.     

Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.