Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.

Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride.

The putative Ca(2+)-channel blocker LaCl3 prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301-310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3 completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl3 inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca(2+)-dependent pathway involving modulation of cytosolic Ca2+ at various stages.

Regulation of the gravitropic response and ethylene biosynthesis in gravistimulated snapdragon spikes by calcium chelators and ethylene inhibitors.

The possible involvement of Ca2+ as a second messenger in snapdragon (Antirrhinum majus L.) shoot gravitropism, as well as the role of ethylene in this bending response, were analyzed in terms of stem curvature and gravity-induced asymmetric ethylene production rates, ethylene-related metabolites, and invertase activity across the stem. Application of Ca2+ chelators (ethylenediaminetetraacetic acid, trans-1,2-cyclohexane dinitro-N,N,N',N'-tetraacetic acid, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid) or a Ca2+ antagonist (LaCl3) to the spikes caused a significant loss of their gravitropic response following horizontal placement. Conversely, the Ca2+ ionophore A23187 or the agonist Bay K-8644 increased gravibending. Longitudinally halved stem sections had significantly higher amounts of ethylene, 1-aminocyclopropane-1-carboxylic acid, and 1-(malonylamino) cyclopropane-1-carboxylic acid compared with vertical controls, with the extra production arising exclusively from the lower half of the stem. trans-1,2-cyclohexane dinitro-N,N,N',N'-tetraacetic acid pretreatment completely abolished the gravity-induced ethylene gradient across the stem, thereby leading to a significant reduction of the curvature. Similarly, reduction of the ethylene produced in the gravistimulated with CoCl2 or inhibition of its action by silver thiosulfate or 2,5-norbornadiene significantly inhibited the subsequent gravibending. Silver thiosulfate and CoCl2 also abolished the gravity-induced gradient of invertase activity across the stem, which is associated with the asymmetric stem elongation. These results suggest that cytosolic Ca2+ may regulate auxin action in snapdragon spikes, manifested as increased ethylene production, which is, in turn, intimately correlated with stem bending. Therefore, both hormones seem to play significant roles in induction and progress of the gravibending of snapdragon spikes.

Chromoplast Biogenesis in Cucumis sativus Corollas (Rapid Effect of Gibberellin A3 on the Accumulation of a Chromoplast-Specific Carotenoid-Associated Protein).

The development of cucumber (Cucumis sativus L.) corollas is accompanied by the accumulation of chromoplasts. In mature corollas, chromoplasts, but no chloroplasts, were detected by electron microscopy. Chlorophyll was also undetectable in corollas at anthesis. The contents of carotenoids and a carotenoid-associated, chromoplast-specific, 35-kD protein in corollas increased in parallel with flower development, peaking concomitantly at anthesis. The involvement of phytohormones and light in the regulation of their expression was studied. When gibberellin A3 (GA3) was added to an in vitro bud culture system, accumulation of both carotenoids and the 35-kD protein was markedly enhanced. The specific up-regulation of the 35-kD protein was very rapid: after only 2 h of culture, increased levels were detected in GA3-treated versus untreated corollas. During this period, corolla fresh weight and total protein and carotenoid contents remained unchanged. Inclusion of abscisic acid in the culture medium counteracted the effect of GA3. Accumulation of the 35-kD protein was also enhanced when flower buds on plants were sprayed with GA3 or etiolated.

Isolation and Characterization of a Chromoplast-Specific Carotenoid-Associated Protein from Cucumis sativus Corollas.

The differentiation of chloroplasts to chromoplasts in corollas of cucumber (Cucumis sativus) is subject to developmental control. To study factors involved in the chloroplast-chromoplast conversion, a chromoplast-specific protein of 35 kD was isolated, and polyclonal antibodies were prepared against it. This protein was found to be a principal component of the carotenoid-protein complex resolved from chromoplast membranes by nondenaturing gel electrophoresis. Immunological studies revealed that expression of this protein is regulated in a temporal and tissue-specific manner. Its steady-state level increased in parallel with flower development and carotenoid accumulation, peaking in mature flowers and then rapidly decreasing to very low levels. The protein was not detectable in cucumber leaves or fruits. To ascertain whether an organ-specific system regulates the chloroplast-chromoplast conversion and to enable future molecular studies of factors involved in this regulation, an in vitro bud culture system was established. Patterns of expression of the 35-kD protein and carotenoids in corollas of detached buds were similar to those in intact buds.

Auxin inhibition of flower induction of pharbitis is not mediated by ethylene.

Treatment of whole Pharbitis nil seedlings or cotyledons with indole butyric acid (IBA) immediately before an inductive dark period greatly inhibited flowering. Treatment of the shoot tip alone with IBA had little or no effect. 1-Aminocyclopropane 1-carboxylic acid, which increased ethylene production by the seedlings much more than IBA, had no effect on the flowering response. Pretreatment of seedlings with the ethylene biosynthesis inhibitor aminooxyacetic acid or with the inhibitor of ethylene action silver thiosulfate did not reduce the inhibitory effect of IBA on flower induction. We concluded, therefore, that the auxin-induced inhibition of flowering of P. nil was not mediated by ethylene.

Stamens and Gibberellic Acid in the Regulation of Flavonoid Gene Expression in the Corolla of Petunia hybrida.

Stamen removal at an early stage of flower development inhibits anthocyanin synthesis and chalcone flavanon isomerase (CHI) enzyme activity in corollas of Petunia hybrida. The inhibition can be overcome by gibberellic acid (GA(3)) application. Gibberellin also induces anthocyanin synthesis in detached, young green corollas, grown in vitro in a sucrose medium and promotes CHI enzyme activity. Western blot analysis indicates an increase in chalcone synthase (CHS) and CHI protein levels following GA(3) treatment in both the in vivo and the in vitro systems. Northern blot analysis shows a higher level of steady-state mRNAs for CHS and CHI 24 hours after GA(3) application. In corollas from a transgenic plant containing a beta-glucuronidase gene driven by a CHI promoter, a sixfold increase of beta-glucuronidase activity was measured following GA(3) application. The mode of action of stamens and GA(3) control over flavonoid gene expression is discussed.

Flowering Response of Pharbitis nil to Agents Affecting Cytoplasmic pH.

Permeant weak acids and auxins have been shown to reduce the cytosplasmic pH in several systems. Lactic, citric, formic, butyric, salicylic, parahydroxybenzoic, propionic acid, and sodium propionate inhibited the flowering response of Pharbitis nil seedlings when applied immediately before an inductive dark period. The acidic auxins IAA, indolebutyric, and alpha-naphtaleneacetic acid, as well as the nonacidic auxin alpha-naphtaleneaceteamid, also inhibited the flowering response. Inhibition was generally more pronounced with a 12-hour than with a 16-hour dark period. Salicylic acid and sodium propionate shifted the response curve of the dark period by about 2 hours. Salicyclic acid, sodium propionate, and indolebutyric acid were inhibitory when applied during the first few hours of the dark period. The permeant weak bases NH(4)Cl, procaine, and trisodium citrate enhanced the flowering response. NH(4)Cl reduced the length of the critical dark period. The inhibition of flowering by acids and auxins as well as the promotion of flowering by bases was obtained even when only the cotyledons had been treated. The inhibition of floral induction by auxins may not be dependent on their effect on the cytoplasmic pH.

Involvement of Calcium in the Photoperiodic Flower Induction Process of Pharbitis nil.

EGTA, a specific Ca(2+) chelator, inhibited the flowering response of Pharbitis nil when applied to the cotyledons immediately before the inductive dark period. Calcium sprayed 30 minutes after the EGTA blocked the effect of EGTA. The length of the critical dark period was increased both by EGTA and by LaCl(3). The calmodulin antagonists W-7 and chlorpromazine also reduced the flowering response. On the other hand, A23187, a calcium ionophore, increased the flowering response. Both EGTA and A23187 were effective at certain times of the photoperiod but had almost no effect when applied at other times. The results indicate that the level of endogenous Ca(2+) may be limiting for floral induction in Ph. nil. Ca(2+) seems to play a role during the early stages of the inductive dark period.

Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hybrida.

Removal of stamens, or even of only the anthers, at an early stage of corolla development, before the start of main anthocyanin production, inhibited both growth and pigmentation of attached corollas of Petunia. When only one or two stamens were removed from one side, the inhibition was restricted to the corolla side adjacent to the detached stamens. Application of gibberellic acid (GA3) substituted for the stamens in its effect on both growth and pigmentation. In detached corollas, isolated at the early-green stage and grown in vitro in sucrose medium, GA3 promoted growth and was essential for anthocyanin synthesis. A marked enhancement of anthocyanin production was observed 48 h before the increase in corolla growth rate. Corollas detached at later stages were able to continue their growth and pigmentation in sucrose without GA3. When Paclobutrazol (ß-[(4-chlorophenyl)-ethyl]-α(1,1-dimethylethyl)-H-1,2,4-triazol-1-ethanol), an inhibitor of gibberellin biosynthesis, was added to the growth medium of in-vitro-grown corollas, pigmentation was inhibited but there was no effect on corolla growth. Low levels of GA3 counteracted the Paclobutrazol effect on pigmentation but did not affect growth. The above results indicate that the effect of GA3 (and probably that of the stamens) on corolla growth is independent of its effect on pigmentation. Gibberellic acid and paclobutrazol had no effect on [(14)C]sucrose uptake by in-vitro-grown corollas. The activity of phenylalanine ammonialyase was correlated with the effect of stamens and GA3 on pigmentation in corollas grown in vivo and in vitro.

Photosynthetic activities in the petunia corolla.

Pink Petuniahybrida (cv Hit Parade Rosa) corollas were found to contain photosynthetically active chloroplasts. The corolla chloroplasts were similar to those of green leaves in size and structure. The chlorophyll (Chl) content of Petunia corollas increased during early stages of flower development, reaching a maximum just before anthesis. Chloroplasts isolated from corollas at this stage, carried out photosystem I-dependent electron transport at rates which were two-thirds of those measured in chloroplasts from green leaves, but full chain electron transport at only one-quarter of the rate carried out by chloroplasts from green leaves. Both the light saturated rate and the quantum yield for electron transport were lower in corolla chloroplasts, which also required lower intensities for light saturation. Reduced efficiency of photosystem II photoreactions in the corolla was also indicated by the ratio between variable and constant components of Chl fluorescence, which was lower in corollas compared to green leaves. The induction time of Chl fluorescence was at least three times shorter in corollas compared to green leaves, indicating a smaller number of functional photosystem II centers (per Chl) in the corolla. It is suggested that corolla chloroplasts of Petunia might have a role in flower developmental processes.

The induction of contractile roots in Gladiolus grandiflorus.

The number of contractile roots formed in gladioli was inversely related to the depth of the planted corm. Below a certain depth, no contractile roots were produced. Large corms did not produce contractile roots at any planting depth but produced two or more small corms. Depth perception is a function of two independent mechanisms, namely, temperature fluctuations in the root-initiation zone and the amount of light perceived mainly by the upper sheath leaf, the length of which varies with planting depth. Various growth substances applied to the leaves or corms did not induce contractile roots in dark-grown plants but roots were induced by indole-3-butyric acid in both small and large corms grown at constant temperatures and light. Abscisic acid retarded the formation of contractile roots under inductive conditions.

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.

Inhibition imposed by developing flowers on further flower-bud initiation in Chamelaucium uncinatum Schau.

In Chamelaucium uncinatum, an Australian woody perennial, flower initiation ceases under continuous inductive short-day (SD) conditions after the first flowering flush. The developing flowers were found to be the prime cause of the cessation in flower initiation. Removal of flowering shoots or lowers as soon as the buds appeared resulted in continuous flower formation. Pruning the plants below the young flower buds at the same stage also caused increased flower formation at the tips of the new growth. If pruning was delayed until flower buds were approx. 3 mm in diameter, however, nor further flower initiation took place and the plants, although still under inductive conditions, shifted to vegetative growth. The inhibiting factor is translocated from one branch to another. At least a six-week "rest" period (a vegetative growth period under long-day conditions) is needed before the plants are able to respond to further SD stimuli.

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 n(6)-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 mul/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.

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.

Senescence and the Fluidity of Rose Petal Membranes : RELATIONSHIP TO PHOSPHOLIPID METABOLISM.

In previous work, senescence of rose petal cells has been shown to be accompanied by a gradual decrease of membrane fluidity, as measured by a fluorescence polarization technique. Concomitantly, an increase in the free sterol-to-phospholipid ratio was found. Both observations were verified in this study. Further, experiments carried out on whole tissue and isolated protoplasts during senescence revealed that there was no quantitative change in the level of free sterols. The content of phospholipids decreased without any significant change in their composition. Results from experiments measuring the incorporation of [(32)P]orthophosphate indicated a reduced capacity for phospholipid synthesis in senescent cells. Both young and old tissue showed phospholipase A and D activity, the former increasing with age.It was concluded that the fluidity of rose petal membranes decreases with age as a result of a decrease in phospholipid content, brought about by both reduced synthesis and enhanced degradation. Evidence supporting the view that the phenomena observed are related specifically to changes in the plasmalemma is discussed.

Promotion of sink activity of developing rose shoots by light.

Holding young rose shoots (Rosa hybrida cv. Marimba) in darkness while the rest of the plant was in light reduced the amount of (14)C assimilates recovered from the darkened shoot by half. Relative specific activity of the shoot tip grown in light was 13.5 times greater than that of the darkened one. The flower bud at the shoot tip degenerated in darkness and died. Shoots 2 to 3 centimeters long, after flower initiation, were most sensitive to the dark treatment. The degeneration is a gradual and reversible process in the first 8 days of darkness, followed by irreversible damage and atrophy. Darkening enhanced the ability of the young leaves to compete for the available assimilates over that of the darkened shoot tip. The enhancement of the mobilizing ability of the shoot tip by light is independent of photosynthesis since spraying with 3-(3,4-dichlorophenyl)-1,1-dimethylurea or holding shoots in a CO(2)-free atmosphere did not diminish the promoting effect of light on flower bud development or assimilate import. The possibility that light exerts its effect by photoproduction of ATP was also excluded inasmuch as no differences were found in ATP levels of shoot tips held in darkness and those held in light.

Characterization of the light reaction in promoting the mobilizing ability of rose shoot tips.

Mixed fluorescent and incandescent light increased growth and sink strength of the uppermost young shoot of rose plants (Rosa hybrida cv. Marimba) in comparison to pure fluorescent light. This was manifested by increased apical dominance. Monochromatic low-energy red light, given by means of optic fibers for 24 hours to shoot tips that had been previously darkened for 5 days, increased the transport of (14)C-labeled assimilates to the intact tips and the uptake of [(14)C]sucrose by detached tips. Far-red had little or no effect, and blue was not effective at all in these reactions. Red light given directly to detached shoot tips, in vitro, increased the uptake of [(14)C]sucrose by the isolated tips. Adding far-red to the red greatly promoted the uptake, whereas blue and blue plus far-red were not active. The main character of the light reaction promoting sink activity in the shoot is that it is perceived by the shoot tip itself. It is operated by red light; far-red promotes the red effect but has little or no effect when alone. Light apparently promotes shoot sink activity by increasing the unloading process.