Gibberellins in the control of photoperiodic flower transition in Pharbitis nil

The role of gibberellins in the photoperiodic flower induction of short-day plant Pharbitis nil has been investigated. It has been found that the endogenous content of gibberellins in the cotyledons of P. nil is low before and after a 16-h-long inductive dark period. During the inductive night the content of gibberellins is high at the beginning of darkness and about the middle of the dark period. Exogenous GA₃ when applied to the cotyledons of non-induced plants does not replace the effect of the inductive night but it can stimulate the intensity of flowering in plants cultivated on suboptimal photoperiods. GA₃ could also reverse the inhibitory effect of end-of-day far-red light irradiation on P. nil flowering. 2-Chloroethyltri-methylammonium chloride (CCC) applied to the cotyledons during the inductive night also inhibited flowering. GA₃ could reverse the inhibitory effect of CCC. The obtained results strongly suggest that gibberellins are involved in the phytochrome controlled transition of P. nil to flowering. Their effect could be additive to that of photoperiodic induction.     

Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil

Hormones are included in the essential elements that control the induction of flowering. Ethylene is thought to be a strong inhibitor of flowering in short day plants (SDPs), whereas the involvement of abscisic acid (ABA) in the regulation of flowering of plants is not well understood. The dual role of ABA in the photoperiodic flower induction of the SDP Pharbitis nil and the interaction between ABA and ethylene were examined in the present experiments. Application of ABA on the cotyledons during the inductive 16-h-long night inhibited flowering. However, ABA application on the cotyledons or the shoot apices during the subinductive 12-h-long night resulted in slight stimulation of flowering. Application of ABA also resulted in enhanced ethylene production. Whereas nordihydroguaiaretic acid (NDGA) - an ABA biosynthesis inhibitor - applied on the cotyledons of 5-d-old seedlings during the inductive night inhibited both the formation of axillary and of terminal flower buds, application of 2-aminoethoxyvinylglycine (AVG) and 2,5-norbornadiene (NBD) - inhibitors of ethylene action - reversed the inhibitory effect of ABA on flowering. ABA levels in the cotyledons of seedlings exposed to a 16-h-long inductive night markedly increased. Such an effect was not observed when the inductive night was interrupted with a 15-min-long red light pulse or when seedlings were treated at the same time with gaseous ethylene during the dark period. Lower levels of ABA were observed in seedlings treated with NDGA during the inductive night. These results may suggest that ABA plays an important role in the photoperiodic induction of flowering in P. nil seedlings, and that the inhibitory effect of ethylene on P. nil flowering inhibition may depend on its influence on the ABA level. A reversal of the inhibitory effect of ethylene on flower induction through a simultaneous treatment of induced seedlings with both ethylene and ABA strongly supports this hypothesis.     

[14C]-Assimilate partitioning in photoperiodically induced seedlings of Pharbitis nil. The effect of benzyladenine

Partitioning of [¹⁴C]-labeled assimilates was studied in relation to photoperiodic floral induction and evocation in one-week-old Pharbitis nil Choisy cv. 'Violet' seedlings. In plants kept under 16 h photoperiods, one 15 h night induced 100% axillary flowering whereas a 24 h night induced both terminal and axillary flowering. A 15 min night break of red light given 8 h after the beginning of the dark period inhibited flowering. Total [¹⁴C]-assimilate distribution among major sinks (plumules + epicotyl and roots + hypocotyl) from a single source cotyledon was unchanged by one inductive night; however, import of [¹⁴C]-assimilates into shoot apices was increased in induced plants compared to vegegative controls. This increase was several-fold in plants subjected to a 24 h night. N⁶-Benzyladenine (BA) application to cotyledons or plumules under non-saturating night lengths increased the number of floral buds per plant without affecting the position of the first floral bud (i.e. the speed of induction). The same treatment caused increased label accumulation in induced apices, while it only slightly affected non-induced ones. The mode of action of BA on flowering through growth stimulation and resulting assimilate mobilization is discussed.     

Flower development in the passion fruit Passiflora edulis requires a photoperiod‐induced systemic graft‐transmissible signal

Different organisms use gradual seasonal changes in photoperiod to correctly time diverse developmental processes, such as transition to flowering in plants. Florigen is a systemic signal formed in leaves exposed to specific environmental cues, mainly photoperiodic, and capable of triggering flower induction in several species. Here we show that in Passiflora edulis, a perennial climbing vine, flower initiation occurs throughout the year; however, without long photoperiods, flower primordia show arrested growth and differentiation at an early stage. Our results support the existence of a positive, systemic, graft‐transmissible signal, produced in mature leaves under LDs, that is required for normal flower development beyond sepal formation. Our results also suggest that Gibberellin acts to inhibit flower development. We provide evidence for genetic variation in the response to short photoperiods. A genotype capable of forming developed flowers under short photoperiods produces a positive graft transmissible signal allowing normal flower development under short days in a cultivar which normally aborts flower development under these conditions. We believe these findings contribute towards discovering the chemical nature of this interesting mobile signal involved in flower development.     

The role of indol-3-ylacetic acid in regulation of juvenility inXanthium strumarium L.

Cotyledons ofXanthium strumarium, organs with low sensitivity to photoperiodic treatment show a higher free indol-3-ylacetic acid level (by about 35 %) than the first pair leaves, organs with high sensitivity to photoperiodic treatment. This was seen in plants of three different age groups : A. with the first pair of leaves of 15–20 mm in length; B. with the first pair of leaves having finished their growth and C. with the third leaf of 30–40 mm in length. Changes in free IAA level during the inductive dark period were similar in both cotyledons and leaves of the first pair. The level of IAA rose in the first half of the dark period, began to decrease in the latter half, reaching nearly initial level at its end. Application of IAA (10⁻⁴ – 10⁻²M) to the cotyledons reduced their already low photoperiodic sensitivity resulting in inhibition of flowering (almost 70 % using 10⁻⁴M IAA). Elevated free IAA level is assumed to be one of the causal factors of low photoperiodic sensitivity of cotyledons.     

Inhibition of stem growth and flower formation in Pharbitis nil with N,N-dimethylaminosuccinamic acid (B 995)

Summary In the short-day plant Pharbitis nil, strain Violet, flower formation is inhibited by application of the growth retardant N,N-dimethylaminosuccinamic acid (B 995) via the roots for a period of 24 hours prior to one inductive long night. Terminal flower bud formation is suppressed by a B 995 concentration of 100 mg/l, but for complete suppression of all axillary flower buds 2000 mg/l is required. Inhibition of flower formation is also caused by B 995 application via plumules or cotyledons, even if made at the end of the inductive night. B 995 treatment always results in short, thick internodes and dark-green leaves.Transport of ¹⁴C-labeled B 995 from cotyledons to plumules and roots takes place during a 16-hour dark period. However, very little label moves from a treated to an untreated cotyledon. Application of B 995 to one of the two cotyledons results in flower inhibition, although the untreated cotyledon produces sufficient flower hormone to induce optimal flower formation. It is concluded therefore that in the short-day plant Pharbitis B 995 does not affect flower hormone production, but rather inhibits floral initiation by interfering with the action of the hormone in the shoot apex.Inhibition of flower formation by B 995 can be completely overcome by application of gibbrellin A₃ to the plumulus before the long nigh. A dose of 0.01 g GA₃/apex is sufficient to re-establish flowering, but much more GA₃ is required to restore internode length equal to that of the control. Indole-3-acetic acid and naphthalene acetic acid are totally inactive in overcoming B 995 inhibition of flower formation and growth.The growth rate of Pharbitis plants treated with B 995 and continuously grown in long-day conditions is initially low, but reaches the same level as in untreated plants approximately 25 days after treatment. ¹⁴C-labeled B 995 applied to cotyledons accumulates to a high degree in roots and in the basal part of the shoots. ¹⁴C-B 995 is metabolized very slowly and persists therefore in Pharbitis plants for prolonged periods of time.     

Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet

Floral induction in seedlings of Pharbitis nil strain Violet, with one cotyledon removed, was manipulated by applying various ethylene treatments to the remaining cotyledon during a 16 hour inductive dark period. Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours at different times during the dark period inhibited flowering to some extent, with inhibition being greater towards the end of the dark period. RNA from cotyledons given a 16 hour dark period (induced) or exposed to 100 microliters per liter ethylene throughout the dark period, which completely inhibited flowering, was examined. The poly(A)⁺RNA was translated in vitro using a wheat germ system, and the resulting translation products were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were substantial qualitative and quantitative differences between the poly(A)⁺RNA extracted from induced cotyledons and that from those exposed to ethylene throughout the dark period. Some of these changes are similar to those observed when flowering was inhibited by photoperiodic treatments (M Lay-Yee, RM Sachs, MS Reid 1987 Planta. In press). The significance of these findings to our understanding of the molecular control of flower induction is discussed.     

Bi-directional inflorescence development inArabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of paraclades

In this study we investigated Arabidopsis thaliana (L.) Heynh. inflorescence development by characterizing morphological changes at the shoot apex during the transition to flowering. Sixteen-hour photoperiods were used to synchronously induce flowering in vegetative plants grown for 30 d in non-inductive 8-h photoperiods. During the first inductive cycle, the shoot apical meristem ceased producing leaf primordia and began to produce flower primordia. The differentiation of paraclades (axillary flowering shoots), however, did not occur until after the initiation of multiple flower primordia from the shoot apical meristem. Paraclades were produced by the basipetal activation of buds from the axils of leaf primordia which had been initiated prior to photoperiodic induction. Concurrent with the activation of paraclades was the partial suppression of paraclade-associated leaf primordia, which became bract leaves. The suppression of bract-leaf primordia and the abrupt initiation of flower primordia during the first inductive photoperiod is indicative of a single phase change during the transition to flowering in photoperiodically induced Arabidopsis. Morphogenetic changes characteristic of the transition to flowering in plants grown continuously in 16-h photoperiods were qualitatively equivalent to the changes observed in plants which were photoperiodically induced after 30 d. These results suggest that Arabidopsis has only two phases of development, a vegetative phase and a reproductive phase; and that the production of flower primordia, the differentiation of paraclades from the axils of pre-existing leaf primordia and the elongation of internodes all occur during the reproductive phase.     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

Root-shoot correlation linked with photoperiodic floral induction inChenopodium rubrum L.

Inhibition of root growth was observed inChenopodium rubrum under photoperiodic conditions inducing flowering. That this inhibition is mediated by the cotyledons was shown directly by the effect of their excision, which changes the responsiveness of the roots to photoperiodic treatment. On the other hand, decapitation did not lead to such an effect. Some evidence is put forward suggesting that changes in IAA may be involved in these correlations. The existence of two different mechanisms of photoperiodic action in flowering and in root growth is proposed to explain these differences.     

Effect of varying lengths of inductive and supplementary non-inductive photoperiods on vegetative growth and flowering of Impatiens balsamina

The photoperiodic requirement for flowering in Impatiens balsaminachanges with the length of the photoperiod. Floral buds wereinitiated with two 8 hr but with four 15 hr photoperiods andflowers opened with four 8 hr but twenty-eight 15 hr photoperiods.A part of the photoperiodic requirement for floral inductionin this plant can be substituted by LDs containing 4 or morehours of darkness (10). It indicates the identical nature ofthe floral stimulus produced during the dark period, whetherit forms a part of the inductive or non-inductive cycles. Theeffect of these supplementary non-inductive photoperiodic cyclesin causing floral bud initiation also depends on the lengthof the first inductive obligatory cycle. More floral buds andflowers were produced on plants exposed to 15 hr than 8 hr photoperiods,probably due to the higher number of leaves that were producedunder the former condition of weaker induction. The shorterthe dark period in the photoperiodic cycle, the weaker the induction,the slower the rate of extension growth but the more differentiationof leaves. ¹ Present address: Department of Biology, Guru Nanak Dev University,Amritsar-143005, India. (Received November 9, 1977; )     

Cytokinins in photoperiodic induction of flowering in Chenopodium species

Changes in cytokinin (zeatin – Z, zeatin riboside – ZR, isopentenyladenine – iP, isopentenyladenosine – iPA) levels were determined under light regimes inductive and non-inductive for flowering in leaves, stems, roots and apical parts of short-day Chenopodium rubrum and long-day Chenopodium murale. In leaves. stems and roots of both plant species the level of cytokinins (in C. rubrum of Z and ZR, in C. murale of Z. ZR, iP and iPA) decreased by about 50% during the dark period and increased again during the subsequent light period, No significant changes in cytokinin levels were observed in continuous light. In apical parts of C. rubrum cytokinin level (Z, ZR, iP) was dramatically increased (by 400–500%) at the end of the dark period and decreased to about the original value during the following light period, while no changes were observed in continuous light. In apical parts of C. murale the level of cytokinins doubled during floral induction consisting of 10 days of continuous light. A red (R) break (15 min at the 6th h of darkness), which prevents flowering in C. rubrum , has no significant effect on cytokinin levels in leaves at the end of darkness. Cytokinin levels increased 1 h after R and decreased again rapidly. On the other hand, the increase of cytokinin level in the apical parts of C. rubrum was largely prevented by the R break. These effects of R on cytokinin levels were not reverted by far-red (FR), while the effect on flowering was reverted. It may be concluded that there is no correlation between changes in cytokinin levels in leaves. Stems and roots and photoperiodic flower induction, as both species, representing different photoperiodic types, showed similar changes under the same light regime. The increase of cytokinin levels in apical parts of both photoperiodic species during floral induction suggests a role (increased cell division and branching) for cytokinins in apex evocation.     

Light Rquirements for Photoperiodic Sensitivity in Cotyledons of Dark-grown Pharbitis nil

The light requirements for induction of flowering by a long dark period were investigated in dark-grown seedlings of Pharbitis nil Chois, cv. Violet. The cotyledons bcame photoperiodically sensitive to a 24 h dark period by two 1 min red irradiations (6.3 μmol m⁻² S⁻¹) separated by a 24 h dark period. The reversibility of the effect of brief red irradiations, and the effectiveness of low energies of red irradiation suggest the involvement of phytochrome in the induction of photoperiodic sensitivity. Partial de-etiolation occurred after these brief periods of red irradiation but the seedlings were not capable of net CO₂ uptakeeven 7 h after the start of the main light period that followed the critical dark period. A changing response to the duration of the priod of darkness given between the two short red irradiations showed the the correct phasing of an endogenous photoperiodic rhythm is needed for the attainment of photoperiodic snsitivity.     

Floral and growth responses in Chenopodium rubrum L. to an extract from flowering Nicotiana tabacum L.

Flowering of Chenopodium rubrum seedling plants was obtained in continuous light after application of fractions of a partially purified extract from leaves of flowering Maryland Mammoth tobacco (Nicotiana tabacum). The stage of flowal differentiation was dependent on the age of the Chenopodium plants used for the bioassay. Apices of plants treated with the extract at the age of four or seven days showed an advanced branching of the meristem or the beginning of formation of a terminal flower; treatment with the extract of plants 12 d old resulted in rapid formation of flower buds in all assay plants. Non-treated control plants kept in continuous light remained fully vegetative. The effects of the extract on flowering were associated with pronounced growth effects. Floral differentiation was preceeded by elongation of the shoot apex. Extension of all axial organs occurred, while growth of leaves, including leaf primordia, was inhibited. The pattern of growth after application of the flower-inducing substance(s) did not resemble the effects of the known phytohormones, but showed some similarities to growth changes resulting from photoperiodic induction of flowering.     

Independent effects of jasmonates and ethylene on inhibition of <Emphasis Type="Italic">Pharbitis nil</Emphasis> flowering

Interactions between methyl jasmonate (JA-Me) and ethylene in the photoperiodic flower induction of short-day plant Pharbitis nil were investigated. Both JA-Me and gaseous ethylene applied during the inductive long night caused a decrease in the number of flower buds generated by P. nil. Application of ethylene did not affected niether the level of endogenous jasmonates in the cotyledons during the 16 h long inductive night, nor the inhibitory effect of JA-Me on the flowering of P. nil accompanied by variations in ethylene production. The application of acetylsalicylic acid (aspirin)—a jasmonate biosynthesis inhibitor—slightly stimulated flowering. Our results have shown that the mechanisms of P. nil flower inhibition by jasmonates and ethylene are independent.     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Changes in the content of endogenous auxins in apical buds ofchenopodium rubrum L. Induced with respect to the endogenous rhythm in capacity to flower

The content of endogenous auxins was examined in apical buds ofChenopodium rubrum plants induced by a photoperiodic cycle of 16h darkness and 8h light followed by a dark period of various duration so as to correspond with either maximal or minimal flowering response in the endogenous rhythm in capacity to flower initiated by the photoperiodic treatment. Apical buds of potentially generative plants contained less auxins than apical buds of plants which remained in the vegetative state. Apical buds from plants treated with kinetin (1. 10^-3 M) and therefore remaining in the vegetative state showed an auxin level comparable to that of untreated plants exhibiting minimal flowering response irrespective of the duration of the second dark period. Plants cultivated on a sucrose solution (0.6 M) during the second dark period became generative even at the normal minimum of flowering. The auxin content of the apical buds was low, similarly as in untreated plants induced for a period leading to maximal flowering response. On the other hand, apical buds from plants grown on sucrose solution during a dark period leading to the manifestation of maximal flowering response showed a relatively high auxin content comparable to that found in untreated plants which had obtained a more extended induction by three photoperiodic cycles. The results are discussed with respect to the possible role of endogenous auxins in the regulation of the changes in growth correlations occurring in the shoot apex during photoperiodic induction and in the expression of the competence to flower.     

Exogenously applied melatonin (N-acetyl-5-methoxytryptamine) affects flowering of the short-day plant Chenopodium rubrum

Melatonin ( N -acetyl-5-methoxytryptamine) is an animal hormone synthesized predominantly at night. It often serves as a signal of darkness that regulates circadian rhythmicity and photoperiodism. Melatonin has also been found in algae and higher plants, including the short-day flowering plant Chenopodium rubrum . To test its involvement in plant photoperiodism, melatonin solutions were applied to the cotyledons and plumules of 5-day-old-seedlings of Chenopodium rubrum L., ecotype 374. ³H-labelled melatonin was readily taken up by the plants and was very stable for a period of 37 h from application. Treatment with 100 and 500 µ M melatonin significantly reduced flowering of plants exposed to a single inductive 12-h darkness. Melatonin was efficient only when applied before lights off or during the first half of the dark period. This indicates that melatonin affects some early steps of the transition to flowering. However, it had no effect on the period or phase of a circadian rhythm in photoperiodic time measurement. Melatonin agonists (2-I-melatonin, 6-Cl-melatonin, CGP 52608) and 5-hydroxytryptamine also reduced flowering, whereas 5-methoxytryptamine did not. The results demonstrate that exogenous melatonin is able to influence the early stages of photoperiodic flower induction and/or flower development in a higher plant. Possible mechanisms for this effect are discussed.     

Effect of Gibberellic Acid and Phosfon on the Critical Dark Period Reqnirement for Flowering of Impatiens balsamina cv. Rose

The critical dark period requirement for flowering of Impatiens balsamina L. cv. Rose, an obligate short day plant, is about 8.5 hours. While GA₃ completely substituted for the dark period requirement, Phosfon prolonged it to 9.5 hours. GA₃ hastened and Phosfon delayed the initiation of floral buds under all photoperiods. Floral buds opened into flowers only during 8 and 14 hour photoperiods in control and Phosfon-treated plants but during all photoperiods in GA₃-treated ones. The delay in floral bud initiation and flowering was correlated with shifting up of the node bearing the first floral bud and flower respectively. While GA₃ increased the numher of floral buds and flowers in all photoperiods except 8-hour, Phosfon increased their number in the 14-hour photoperiod only. The number of flowering plants decreased with increasing photoperiod regardless of GA₃ and Phosfon application. The effect of Phosfon was completely or partially overcome, depending upon the photoperiod, by simultaneous application of GA₃.