Acyl chain and head group regulation of phospholipid catabolism in senescing carnation flowers

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.     

Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.     

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.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Senescence in isolated carnation petals : effects of indoleacetic Acid and inhibitors of protein synthesis

Indoleacetic acid induces senescence in isolated carnation (Dianthus caryophyllus, cv. White Sim) petals, increasing the duration and amount of ethylene production. This effect is inhibited by Actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, a translational inhibitor of protein synthesis. The ability of petals to respond to indoleacetic acid appears to be a function of physiological age. Indoleacetic acid is capable of enhancing ethylene evolution and senescence only in specific portions of the petal.     

Invertase and sucrose synthase in flowers

Sucrose and reducing sugar concentrations in petals of cut carnation flowers, whose life was prolonged up to 7 days by bathing stalks in sucrose solutions, were respectively 3-fold and 2-fold higher than those bathed in water. Reducing sugar concentrations were about 7-fold higher than sucrose concentrations. A study of invertase and sucrose synthase activities in flower petals of carnation and four other species of flowers revealed that both enzymes may be involved in hydrolysis of translocated sucrose. Invertase activity, while being up to 20-fold higher than sucrose synthase activity in some species was approximately comparable in others. More detailed studies on invertase from petals of 3 flower species demonstrated the presence of only the acid form of the enzyme with a K_m value for sucrose of about 2.5 mM.     

Inhibition of ethylene biosynthesis in carnation petals by cytokinin

Pretreatment of detached carnation petals (Dianthus caryophyllus cv White Sim) for 24 hours with 0.1 millimolar of the cytokinins n⁶-benzyl-adenine (BA), kinetin, and zeatin blocked the conversion of externally supplied 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene and delayed petal senescence by 8 days. The normal enhanced wilting and increase in endogenous levels of ACC and ethylene production following exposure of petals to ethylene (16 μl/l for 10 hours), were not observed in BA-pretreated petals. In carnation foliage leaves pretreated with 0.1 mm BA, a reduction rather than inhibition of the conversion of exogenous ACC to ethylene was observed. This indicates that foliage leaves respond to cytokinins in a different way than petals. A constant 24-hour treatment with BA (0.1 mm) was not able to reduce ethylene production of senescing carnation petals, while 2 mm aminoxyacetic acid, a known inhibitor of ACC synthesis, or 10 mm propyl gallate, a free radical scavenger, decreased ethylene production significantly.     

A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress

The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (−28 bar) or 428 millimolar NaCl (−23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl₂ extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.     

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.     

Respiratory Shifts in Developing Petals of Saxifraga cernua

Changes in the oxygen uptake of petal slices by the cytochrome and alternative respiratory pathways were monitored during petal development in the arctic herb Saxifraga cernua. As the petals developed, rates of total respiration increased to a maximum rate during petal unfolding (day 4.5), and thereafter declined. Respiration in petals of all ages was at least partially resistant to cyanide, indicating the capacity for the alternative pathway. In all, except day 1 and senescing day 8 petals, respiration was inhibited by salicylhydroxamic acid, indicating engagement of the alternative pathway. In general, temporal changes in the respiratory activity along each pathway were similar and in parallel with changes in total respiration, although maximum rates along each pathway occurred at different times. Maximum cytochrome pathway activity occurred during petal expansion (day 4) whereas the alternative pathway peaked during petal unfolding at day 4.5. The control of respiration was also investigated. In the presence of salicylhydroxamic acid, the addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone was never stimulatory, suggesting that the cytochrome pathway was not restricted by adenylate levels. The addition of sucrose stimulated respiration only in day 1 petals, suggesting substrate limitation at this developmental stage. Since the rate of alternative pathway respiration peaked during petal unfolding, a time of high energy requirement, we suggest that the alternative pathway may have been used as an inefficient energy source during petal development.     

Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene

The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1-aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1-carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric.     

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.     

Plastid Ontogeny during Petal Development in Arabidopsis

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.     

1-aminocyclopropane-l-carboxylic acid (ACC)—The transmitted stimulus in pollinated flowers?

Ethylene production and senescence of petals of pollinated carnation flowers were not prevented by removal of the ethylene produced by the gynoecium, suggesting that these events are a response to movement from the gynoecium of some stimulus other than ethylene gas. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to the stigmas caused an initial increase in gynoecium and petal ethylene production similar to that reported for pollinated flowers. This response was not seen in flowers whose stigmas were treated with indoleacetic acid (IAA). When [2-¹⁴C]ACC was applied to the stigmas of carnation flowers, radioactive ethylene was produced both by the gynoecia and by the petals. The possibility that ACC, transported from the stigmas to the petals, is responsible for the postpollination changes in carnation flowers is discussed.     

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.     

Age-Dependent Discrimination between Stereoisomers of 1-Amino-2-Ethylcyclopropane-1-Carboxylic Acid in Carnation Petals

The ability of carnation petals (Dianthus caryophyllus L. cv White Sim) of different ages to convert the cis and trans isomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) to 1-butene was studied. Young petals, which produce ethylene at a low rate, convert both cis- and trans-AEC to 1-butene with low efficiency and at equal rates. In senescing petals, the rate of conversion of cis-AEC remains low, but there is a marked increase in the rate of trans-AEC conversion. Thus there is a clear evidence of stereodiscrimination between the isomers. Stimulating the rate of senescence by treatment with either 1-aminocyclopropane-1-carboxylic acid or ethylene further increases the rate of trans-AEC conversion. Delaying of petal senescence by silver thiosulphate or aminooxyacetic acid inhibits the rise in trans-AEC conversion.