The Involvement of Cyclic ADPR in Photoperiodic Flower Induction of Pharbitis nil

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from NAD⁺ by ADP-ribosyl cyclases described for several animal cells. Pharmacological studies suggest that cADPR is an endogenous modulator of Ca²⁺-induced Ca²⁺ release channels. There is also information about the sub-micromolar concentration of cADPR in plant cells. Whether cADPR can act as a Ca²⁺-mobilizing intracellular messenger in plant tissue is an unresolved question. Despite the obvious importance of monitoring cADPR cellular levels under various physiological conditions in plants, its measurement has been technically difficult and requires specialized reagents. In the present study a widely applicable sensitivity assay for cADPR is described. We show that Pharbitis nil tissue from cotyledons contains a certain cADPR level. To explain the possible roles of this second messenger in photoperiodic flower induction, some physiological experiments were also performed. The exogenous applications of cADPR to Pharbitis nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. Nevertheless 8-Br-cADPR inhibited flowering when these compounds were applied during a 16-h-long inductive night. The effect of ruthenium red, a calcium channel blocker and ryanodine, a calcium channel stimulator, on the photoperiodic induction of flowering was also studied. Ruthenium red, when applied before and during an inductive 16-h dark period, slightly inhibited flowering, whereas ryanodine, when applied before and during a 12-h long subinductive night, stimulated flower bud formation. We also confirmed evidence that Ca²⁺ ions are involved in the photoperiodic induction of flowering. Thus, the obtained results may suggest the involvement of cyclic ADPR-activated Ca²⁺ mobilization in the photoperiodic flower induction process in Pharbitis nil.     

Ethylene and IAA interactions in the inhibition of photoperiodic flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It has been shown that both IAA and ethylene application inhibit flower induction in the short-day plant Pharbitis nil. However application of IAA has elevated ethylene production in this plant, as well. Strong enhancement of ethylene production is also correlated with the night-break effect, which completely inhibits flowering. In order to determine what the role of IAA and ethylene is in the photoperiodic flower induction in Pharbitis nil, we measured changes in their levels during inductive and non-inductive photoperiods, and the effects of ethylene biosynthesis and action inhibitors on inhibition of flowering by IAA. Our results have shown that the inhibitory effect of IAA on Pharbitis nil flowering is not physiological but is connected with its effect on ethylene biosynthesis.     

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.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

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.     

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.     

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.     

Inhibitory Effect of Brassinosteroids on the Flowering of the Short-Day Plant Pharbitis nil

The effect of exogenous brassinosteroids (BR) on the flowering induction of Pharbitis nil was examined. Generally plants treated with brassinolide and castasterone form less number of flowers than control plants, but degree of flowering inhibition was depended on the concentration and the method of BR application as well as the length of the inductive dark period. In plants regenerated from sub-induced apices treated with brassinolide at concentration of 1 and 10 M the flower formation was inhibited completely.     

Different circadian rhythms regulate photoperiodic flowering response and leaf movement in Pharbitis nil (L.) Choisy

The phase shifting effect of red light on both the leaf movement rhythm, and on the rhythm of responsiveness of photoperiodic flower induction towards short light breaks (10 min red light), has been studied in Pharbitis nil, strain Violet, and comparisons between the two rhythms have been made. The phase angle differences between the rhythms after a phase shift with 2 or 6 h of red light given at different times during a long dark period were not constant. The results indicate the involvement of two different clocks controlling leaf movement and photoperiodic flower induction.Abbreviations DD continuous darkness - l:D x:y light/dark cycles with x hours of light and y hours of darkness - PPR rhythm of photoperiodic responsiveness towards light break     

Cyclic GMP stimulates flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis> via its influence on cGMP regulated protein kinase

It was revealed that cGMP is involved in the control of photoperiodic flower induction. Further insight into the signalling function of cGMP is likely to be obtained by analysis of its effectors. Therefore, in the present study, we used various agents that cause changes in cGMP-dependent kinase (PKG) activity and examined their effects on the activity of kinase isolated from Pharbitis nil and flower induction. It was found that exogenous applications of PKG activators (cGMP, 8-pCPT-cGMP, 8-Br-cGMP, 8-pCPT-PET-cGMP) to cotyledons which were exposed to a 12-h-long subinductive night significantly increased flowering response. From among the many antagonists of cGMP-dependent protein kinase Rp-8-Br-PET-cGMPS, Rp-8-pCPT-cGMP and the synthetic heptapeptide inhibitor of PKG were used for our analysis. When Rp-8-Br-PET-cGMPS and Rp-8-pCPT-cGMP were applied during a 16-h-long inductive night, significant reduction in the number of flower buds was observed, whereas synthetic heptapeptide did not change the intensity of flowering. The influence of the analysed chemicals on protein kinase activity was also examined in vitro. With the exception of synthetic heptapeptide, which seems ineffective, the enzyme activity was stimulated by all agonists and significantly reduced by all antagonists. The activity of protein kinase was assayed in P. nil soluble protein fractions from plants grown under flower-inducing and non-inducing conditions. In vitro phosphorylation was slightly greater in the soluble fraction obtained from plants grown under the flower-inducing condition, reaching 1.05 nmol/min/mg protein, when compared to the control 0.81 nmol/min/mg protein. In relation to the results described above, we can conclude that cGMP as a mediator participating in photoperiodic flower induction may govern this process by the phosphorylation mechanism via its influence on cGMP-dependent protein kinase activity.     

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.     

Evidence for the Involvement of Calcium Ions in the Photoperiodic Induction of Flowering in Pharbitis nil

The effects of ethyleneglycol-bis-(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid, a specific chelator of Ca²⁺ ions;lanthanum chloride, a calcium channel blocker; and chlorpromazine,a calmodulin antagonist, on the photoperiodic induction of floweringin Pharbitis nil were studied by perfusing the plants with aqueoussolutions of the various compounds. All these compounds inhibitedflowering when applied before an inductive 16-h dark periodbut they did not inhibit flowering when applied after the inductivedark period. The results imply that Ca²⁺ ions are involved inthe photoperiodic induction of flowering in P. nil. (Received August 14, 1992; Accepted November 24, 1992)     

Changes in organ growth ofChenopodium rubrum due to suboptimal and multiple photoperiodic cycles with and without flowering effect

The growth changes of cotyledons, leaves, hypocotyls and roots due to photoperiodic induction in short day plantChenopodium rubrum were investigated in relation to flowering. Six-day old plants were induced by photoperiods with a different number of dark hours. We found that the degree of inhibition which occurred during induction in the growth of leaves, cotyledons and roots similarly as the stimulation of hypocotyl is proportional to the length of dark period. The photoperiods with 12, 16 and 20 dark hours bring about marked inhibition of growth and at the same time induce flowering in terminal and axillary meristems. The inhibitory effect of critical period for flowering,i.e. 8 dark hours, is not apparent in all criteria used and even the flower differentiation is retarded. The photoperiods of 4 and 6 dark hours did not affect growth and were ineffective in inducing flowering even if their number has been increased. The experiments with inductive photoperiod interrupted by light break have clearly shown that growth pattern characteristic for induced plants can be evoked in purely vegetative ones. Such statement did not exclude the possible importance of growth inhibition as a modifying factor of flower differentiation. We demonstrated that the early events of flower bud differentiation are accompanied by stimulation of leaf growth. The evaluation of growth and development of axillary buds at different nodes of insertion enabled us to quantify the photoperiodic effect and to detect the effects due to differences in dark period length not exceeding 2 hours.     

Two <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in <Emphasis Type="Italic">Chenopodium rubrum</Emphasis> differ in expression patterns

FLOWERING LOCUS T (FT) like genes are crucial regulators (both positive and negative) of flowering in angiosperms. We identified two FT homologs in Chenopodium rubrum, a short-day species used as a model plant for the studies of photoperiodic flower induction. We found that CrFTL1 gene was highly inducible by a 12-h dark period, which in turn induced flowering. On the other hand, photoperiodic treatments that did not induce flowering (short dark periods, or a permissive darkness interrupted by a night break) caused only a slight increase in CrFTL1 mRNA level. We demonstrated diurnal oscillation of CrFTL1 expression with peaks in the middle of a light period. The oscillation persisted under constant darkness. Unlike FT homologs in rice and Pharbitis, the CrFTL1 expression under constant darkness was very low. The CrFTL2 gene showed constitutive expression. We suggest that the CrFTL1 gene may play a role as a floral regulator, but the function of CrFTL2 remains unknown.     

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.     

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.     

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.