The metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil

We examined the involvement of chlorogenic acid (CGA) and salicylic acid (SA) in the stress-induced flowering of Pharbitis nil (synonym Ipomoea nil). The incorporation efficiency of exogenously applied CGA and the deactivation rate of incorporated CGA were determined in cotyledons by high-performance liquid chromatography. The assay plants could not incorporate a sufficient amount of CGA via roots. The perfusion technique by which the assay solution was forced into the plant from the cut end of the hypocotyl improved the efficiency of CGA incorporation. However, no flower-inducing activity was detected, indicating that CGA was not involved in flowering. It was concluded that the close correlation between CGA content and flowering response is merely coincidence or a parallelism. Flowering under long-day conditions induced by low-temperature stress was completely inhibited by aminooxyacetic acid (AOA), an inhibitor of phenylalanine ammonialyase. The flower-inhibiting effect of AOA was nullified by co-applied t-cinnamic acid and by benzoic acid. This indicates that the metabolic pathway from t-cinnamic acid to SA via benzoic acid is involved in the stress-induced flowering. The results indicate that the metabolic pathway of SA is involved in the stress-induced flowering of P. nil not the metabolic pathway of CGA.     

Involvement of cyclic GMP in phytochrome-controlled flowering of Pharbitis nil

Light is one of the most important environmental factors influencing the induction of flowering in plants. Light is absorbed by specific photoreceptors – the phytochromes and cryptochromes system – which fulfil a sensory and a regulatory function in the process. The absorption of light by phytochromes initiates a cascade of related biochemical events in responsive cells, and subsequently changes plant growth and development.

Induction of flowering is controlled by several paths. One is triggered by the guanosine-3′:5′-cyclic monophosphate (cGMP) level. Thus, the aim of our study was to investigate the role of cGMP in phytochrome-controlled flowering.

It is best to conduct such research on short-day plants because the photoperiodic reactions of only these plants are totally unequivocal. The most commonly used plant is the model short-day plant Pharbitis nil.

The seedlings of P. nil were cultivated under special photoperiodic conditions: 72-h-long darkness, 24-h-long white light with low intensity and 24-h-long inductive night. Such light conditions cause a degradation of the light-labile phytochrome. Far red (FR) treatment before night causes inactivation of the remaining light-stable phytochrome. During the 24-h-long inductive darkness period, the total amount of cGMP in cotyledons underwent fluctuations, with maxima at the 4th, 8th and 14th hours. When plants were treated with FR before the long night, fluctuations were not observed. A red light pulse given after FR treatment could reverse the effect induced by FR, and the oscillation in the cGMP level was observed again.

Because the intracellular level of cGMP is controlled by the opposite action of guanylyl cyclases (GCs) and phosphodiesterases (PDEs), we first tested whether accumulation of the nucleotide in P. nil tissue may be changed after treatment with a GC stimulator or PDE inhibitor.

Accumulation of the nucleotide in P. nil cotyledons treated with a stimulator of cGMP synthesis (sodium nitroprusside) was markedly (approximately 80%) higher. It was highest in the presence of dipyridamole, whereas 3-isobutyl-1-methylxanthine did not significantly affect cGMP level.

These results show that the analysed compounds were able to penetrate the cotyledons’ tissue, and that they influenced enzyme activity and cGMP accumulation.

FR light applied at the end of the 24-h-long white light period inhibited flowering. Exogenous cGMP added on cotyledons could reverse the effect of FR, especially when the compound was applied in the first half of the long night. Flowering was also promoted by exogenous application of guanylyl cyclase activator and phosphodiesterase inhibitors, and in particular dipyridamole.

The results obtained suggest that an endogenous cGMP system could participate in the mechanism of a phytochrome-controlled flowering in P. nil.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Effects of abscisic acid on flowering in Pharbitis nil

Under continuous light, flowering of Pharbitis seedlings wasnot induced by abscisic acid (ABA) treatment. Under short daytreatment, flowering was slightly enhanced by ABA at 0.2 and0.4 mg/liter. Stem elongation was considerably inhibited by25 and 50 mg/liter of ABA irrespective of day length. (Received October 21, 1972; )     

The metabolism of inhibitor of flowering and prostaglandin biosynthesis, acetylsalicylic acid, in Pharbitis nil cotyledons

Acetylsalicylic acid, which applied to cotyledons of the short day plant Pharbitis nil prior to an inductive 16-h dark period inhibits flowering by 90 %, is converted to salicylic acid and to a lesser extent to gentisic acid in the cotyledons during this 16-h dark period. Our results confirmed that salicylic acid and gentisic acid are responsible for the inhibition of flowering. They also inhibit prostaglandin biosynthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Benzoic acid may act as the functional group in salicylic acid and derivatives in the induction of multiple stress tolerance in plants

Benzoic acid, sulfosalicylic acid and methyl salicylic acid wereevaluated for their regulatory role in inducing multiple stress tolerance inbean (Phaseolus vulgaris cv Brown Beauty) and tomato(Lycopersicum esculentum cv Romano) plants. All threemolecules were effective in inducing tolerance to heat, drought and chillingstress similar to that reported previously for salicylic and acetylsalicylicacids. Benzoic acid is effective at lower concentrations than salicylic acid orits derivatives. The benzoic acid structural portion is common to all fivemolecules and is the most likely basic functional molecular structure impartingstress tolerance in plants.     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C₈ Nucleosil column (5 μm, 250×4.6 mm) using a mobile phase containing a mixture of water–acetonitrile–orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2–5.0 μg/ml and had a limit of detection of 0.05 μg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis

The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.     

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.     

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures.     

Inhibition of flowering and growth in Pharbitis nil by the growth retardant Ancymidol

Flower formation and growth of the short day plant Pharbitisnil, strain "Violet", were inhibited when the growth retardantAncymidol was applied prior to an inductive dark period viacotyledons or roots. Inhibition of flower formation by Ancymidolcould be completely reversed by an application of gibberellinA₃ (GA₃) to the plumule before the inductive dark period. Adose of 0.01 µg GA₃/plant was almost sufficient to restoreflowering, but about a hundred times more GA₃ was required torestore the internode length to that of control. Ancimidol greatlyreduced the endogenous gibberellin content. (Received July 18, 1980; )     

Inverse correlation between jasmonic acid and salicylic acid during early wound response in rice

This study presents a kinetic analysis of the response to wounding in rice plants. In particular, jasmonic acid, salicylic acid, and lipoxygenase activity were measured in leaves of wounded rice plants during the early tillering phase. The results show that endogenous jasmonic acid transiently increases to a maximum 30 min after wounding (jasmonic acid burst) and lipoxygenase activity increases after the jasmonic acid burst, but not after the second smaller peak of endogenous jasmonic acid 23 h after wounding. In contrast, endogenous salicylic acid decreases during the jasmonic acid burst, such that the kinetic profiles of jasmonic acid and salicylic acid are inversely correlated during the early response to wounding. It is proposed here that the increase in endogenous jasmonic acid and the decrease in endogenous salicylic acid may contribute for establishing the efficient negative cross-talk between jasmonic acid and salicylic acid signaling pathways during the early response to wounding in rice.     

Further studies on the flowering ofLemna paucicostata in Japan

Flowering behavior of 22 strains ofLemna paucicostata collected in Japan by Yukawa and Takimoto (1976) was re-examined. The critical dark periods of the short-day strains (N-1 and N-2 types) were shorter than those determined by Yukawa and Takimoto except for that of one strain. Particularly in strains 391, 381 and 321, the differences were as large as 2.25, 1.75 and 1.5 hr, respectively. Such differences were found to be due at least partly to the difference in night temperature; 25 C for the light and 23 C for the dark periods in the present experiment, and 25 C throughout the light and dark periods in the previous experiment. The S type strains did not flower under our experimental conditions (fluorescent light of 6,000 lux at 25 C) at any photoperiod tested, but flowered as a quantitative long-day plant under natural daylight or high-intensity light (12,000 lux). Addition of sucrose or ammonium ion to the medium suppressed the flowering of these strains under high-intensity light. Addition of benzoic acid (1–5 μM) to 0.5 strength NH₄ ⁺-free Hutner's medium caused daylength-independent flowering in some N-1 type strains and in all N-2 type strains tested. S type strains cultured under fluorescent light of 6,000 lux also flowered rapidly in response to benzoic acid.     

Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.     

Influences of plant hormones on photoperiodic flowering in Pharbitis nil: re-evaluation by the perfusion technique

Influences of plant hormones on photoperiodic flowering in Pharbitis nil, var. Violet was re-evaluated by assaying them with a newly developed perfusion technique which can directly treat mesophyll cells with sample solution. Gibberellin A₃ promoted the flowering response and indole-3-acetic acid, trans-zeatin and abscisic acid inhibited it when they were perfused immediately before an inductive dark treatment. The promotion or inhibition of flowering was not or hardly observed when solutions containing these plant hormones were applied by the dropping method to surface of cotyledons or plumules of the assay plants. The detection of clear flower-promoting and -inhibiting effects of the plant hormones may be due to the improved efficiency of incorporation of applied substances into plant tissue in the perfusion technique.     

Phaseshifting of the photoperiodic flowering response rhythm in Pharbitis nil by red-light pulses

The control by light of the flowering response rhythm in the short-day plant Pharbitis nil Choisy cv. Violet was examined by giving a single pulse of light at various times between 1 and 6 h after a 24-h light period. When the first circadian cycle of the rhythm was monitored, it was found that a pulse of red light given at 1, 2 or 3 h into a 72-dark period caused a 1-h delay of the phase of the response rhythm, while a pulse at 6 h caused a 2-h delay. These results support the hypothesis that, when red-light pulses are given at hourly intervals, they are as effective as continuous light in preventing the onset of dark timing because they repeatedly return the rhythm to the circadian time at which it is apparently suspended in continuous light. The perception of and response to continuous light and red-light pulses are also briefly discussed.     

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.