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.     

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.     

Involvement of Abscisic Acid and Indole-3-acetic Acid in the Flowering of Pharbitis nil

The involvement of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the regulation of flowering of Pharbitis nil was investigated through exogenous applications and analyses of endogenous levels. Both hormones inhibited the flowering of P. nil when they were applied before or after a single 15-h dark treatment. The inhibitory effect of ABA and IAA was significant when they were applied before the dark treatment, and the application to plumules was more effective than that to cotyledons. In all applications, the inhibitory effect of IAA was stronger than that of ABA. Endogenous levels of ABA and IAA in the plumules were compared between flower-inductive (15-h dark treatment) and noninductive (continuous light) light conditions. There was no significant difference in the ABA level between light and dark conditions, whereas the level of IAA was decreased by the dark treatment. These results suggest that biosynthesis and/or catabolism of IAA is affected by the light treatment and therefore may be involved in the regulation of early flowering processes in the apex. The inhibitory effects of ABA and IAA were reversed by an application of gibberellin A₃, indicating that gibberellin A₃ counteracts the flowering processes affected by ABA and IAA. Application of aminoethoxyvinylglycine restored the flowering response inhibited by IAA, which suggests the possibility that the inhibitory effect of IAA is the result of enhanced ethylene biosynthesis. Received November 22, 1996; accepted February 17, 1997     

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.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

EGTA inhibits floral induction of Pharbitis nil via its influence on gas exchange properties of stomata

The purpose of the study was to determine inhibitory effect of calcium chelator; ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) on flowering of a short-day (SD) plant Pharbitis nil. It was found that 20 mM solution of EGTA applied on cotyledons of 5-d-old P. nil seedlings four hours before the start of 16-h-long induction night decreased the flowering response by 55% compared to the control plants not treated with this Ca²⁺ chelator. It also caused a very significant decrease of photosynthesis rate, transpiration rate and stomatal conductance both in light and darkness conditions. The results of this study confirm earlier hypothesis suggesting the effect of Ca²⁺ and its modulators on P. nil flowering is due to their influence on the stomata.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.     

Cross talk between phytohormones in the regulation of flower induction in Pharbitis nil

Application of gibberellic acid (GA₃) on the cotyledons of 5-d-old Pharbitis nil reversed the inhibitory effect of both abscisic acid (ABA) and ethylene on flowering. Application of GA₃ slightly decreased ethylene production and did not affect the endogenous ABA content in the cotyledons during the night. However, it reversed the stimulating effect of ABA on ethylene production.     

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.     

NADP-Dependent Phenylacetaldehyde Dehydrogenase for Degradation of Phenylethylamine in Arthrobacter globiformis

The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA₁ ≧GA₂₀>GA₁₉≧GA₄₄>GA₅₃»GA_H. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.     

Guanylyl cyclase activity during photoperiodic flower induction in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.     

Increase in the Activity of Phenylalanine Ammonia-Lyase during the Non-Photoperiodic Induction of Flowering in Seedlings of Morning Glory (Pharbitis nil)

The activity of phenylalanine ammonia-lyase in the seedlingsof Pharbitis nil strains ‘Violet’ and ‘Kidachi’during the non-photoperiodic induction of flowering increasedin close association with the accumulation of phenylpropanoidsand the induction of flower buds. This observation suggestedthat the increased activity of phenyalanine ammonia-lyase causedthe accumulation of the phenylpropanoids and that the activationof the biosynthesis of phenylpropanoids might be involved inthe non-photoperiodic induction of flowering in the seedlingsof ‘Violet’ and ‘Kidachi’. (Received September 6, 1994; Accepted December 15, 1994)     

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.     

Shoot inversion-induced ethylene production: a general phenomenon?

Shoot inversion induction of ethylene production was found in inverted shoots of corn, peas, soybean, sunflower, tomato, andPharbitis nil. The increases in ethylene production were found to range from two- to threefold in soybean to eightfold in corn and sunflower. The occurrence of peaks of ethylene production ranged from 16 h following shoot inversion in corn to 72 h inPharbitis. That the enhanced ethylene production was due to activation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase is supported by the finding of increased ACC content in inverted shoots of all species tested. Shoot inversion inhibition of elongation was found in inverted shoots of pea, soybean, sunflower, tomato, andPharbitis nil. This inhibition is thought to be mediated via increased ethylene production in the inverted shoots. That shoot inversion induction of ethylene is not a persistent effect is supported by the finding that ethylene synthesis could be terminated by reorientation of shoots to the upright position and could be reinitiated by the subsequent inversion of the shoots. The effects of shoot inversion on the enhancement of ethylene production and on the inhibition of elongation of the inverted shoot appear to be general phenomena.     

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.