Endogenous factors regulating poor-nutrition stress-induced flowering in pharbitis: The involvement of metabolic pathways regulated by aminooxyacetic acid.

The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering.

Climatic factors influence leaf structure and thereby affect the ozone sensitivity of Ipomoea nil 'Scarlet O'Hara'.

Phenotypic plasticity of the leaves can interfere with the plant sensitivity to ozone (O3) toxic effect. This study aimed to assess whether the leaf structure of Ipomoea nil changes due to climatic variations and whether these changes affect the species' sensitivity. Field exposures, in different seasons (winter and spring) were made. The leaves that developed during the winter were thinner, with a lower proportion of photosynthetic tissues, higher proportion of intercellular spaces and lower density and stomatal index compared to those developed during the spring. The temperature and relative humidity positively influenced the leaf thickness and stomatal index. The visible injuries during winter were positively correlated with the palisade parenchyma thickness and negatively correlated with the percentage of spongy parenchyma; during the spring, the symptoms were positively correlated with the stomatal density. In conclusion, the leaf structure of I. nil varied among the seasons, interfering in its sensitivity to O3.

Identification of a NAC transcription factor, EPHEMERAL1, that controls petal senescence in Japanese morning glory.

In flowering plants, floral longevity is species-specific and is closely linked to reproductive strategy; petal senescence, a type of programmed cell death (PCD), is a highly regulated developmental process. However, little is known about regulatory pathways for cell death in petal senescence, which is developmentally controlled in an age-dependent manner. Here, we show that a NAC transcription factor, designated EPHEMERAL1 (EPH1), positively regulates PCD during petal senescence in the ephemeral flowers of Japanese morning glory (Ipomoea nil). EPH1 expression is induced independently of ethylene signaling, and suppression of EPH1 resulted in Japanese morning glory flowers that are in bloom until the second day. The suppressed expression of EPH1 delays progression of PCD, possibly through suppression of the expression of PCD-related genes, including genes for plant caspase and autophagy in the petals. Our data further suggest that EPH1 is involved in the regulation of ethylene-accelerated petal senescence. In this study, we identified a key regulator of PCD in petal senescence, which will facilitate further elucidation of the regulatory network of petal senescence.

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.

Stress and salicylic acid induce the expression of PnFT2 in the regulation of the stress-induced flowering of Pharbitis nil.

Poor nutrition and low temperature stress treatments induced flowering in the Japanese morning glory Pharbitis nil (synonym Ipomoea nil) cv. Violet. The expression of PnFT2, one of two homologs of the floral pathway integrator gene FLOWERING LOCUS T (FT), was induced by stress, whereas the expression of both PnFT1 and PnFT2 was induced by a short-day treatment. There was no positive correlation between the flowering response and the homolog expression of another floral pathway integrator gene SUPPRESSOR OF OVEREXPRESSION OF CO1 and genes upstream of PnFT, such as CONSTANS. In another cultivar, Tendan, flowering and PnFT2 expression were not induced by poor nutrition stress. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase inhibitor, inhibited the flowering and PnFT2 expression induced by poor nutrition stress in Violet. Salicylic acid (SA) eliminated the inhibitory effects of AOA. SA enhanced PnFT2 expression under the poor nutrition stress but not under non-stress conditions. These results suggest that SA induces PnFT2 expression, which in turn induces flowering; SA on its own, however, may not be sufficient for induction.

Critical analysis of the potential of Ipomoea nil'Scarlet O'Hara' for ozone biomonitoring in the sub-tropics.

This study aimed to analyze critically the potential of Ipomoea nil'Scarlet O'Hara' for O(3) biomonitoring in the sub-tropics. Four field experiments (one in each season of 2006) were carried out in a location of the city of São Paulo mainly polluted by O(3). Each experiment started with 50 plants, and lasted 28 days. Sub-lots of five plants were taken at intervals between three or four days long. Groups of four plants were also exposed in closed chambers to filtered air or to 40, 50 or 80 ppb of O(3) for three consecutive hours a day for six days. The percentage of leaf injury (interveinal chloroses and necroses), the concentrations of ascorbic acid (AA) and the activity of superoxide dismutase (SOD) and peroxidases (POD) were determined in the 5th, 6th and 7th oldest leaves on the main stem of the plants taken in all experiments. Visible injury occurred in the plants from all experiments. Seasonality in the antioxidant responses observed in plants grown under field conditions was associated with meteorological variables and ozone concentrations five days before leaf analyses. The highest levels of antioxidants occurred during the spring. The percentage of leaf injury was explained (R(2) = 0.97, p < 0.01) by the reduction in the levels of AA and activity of POD five days before the leaf analyses and by the reduction in the levels of particulate matter, and enhancement of temperature and global radiation 10 days before this same day. Although I. nil may be employed for qualitative O(3) biomonitoring, its efficiency for quantitative biomonitoring in the sub-tropics may be compromised, depending on how intense the oxidative power of the environment is.

The redox state of Ipomoea nil 'Scarlet O'Hara' growing under ozone in a subtropical area.

The occurrence of visible leaf injury caused by ozone in Ipomoea nil 'Scarlet O'Hara' may be regulated by their redox state, affecting its bioindicator efficiency. Thus, this study aimed to determine whether the redox state of I. nil plants in a subtropical area (São Paulo, SE-Brazil) contaminated by ozone oscillates, and to identify the environmental factors behind these variations. We comparatively evaluated indicators of redox state (ascorbic acid, glutathione, superoxide dismutase, ascorbate peroxidase, glutathione reductase) and leaf injury during nine field experiments of 28 days each. The variations in the redox indicators were explained by the combined effects of chronic levels of ozone and meteorological variables (mainly global solar radiation and air temperature) 3-6 days prior to the sampling days. The ascorbic acid and glutathione were crucial for increasing plant tolerance to ozone. Weak visible injury was observed in all experiments and occurred in leaves with low levels of ascorbic and dehydroascorbic acids.

Salicylic acid and the flowering gene FLOWERING LOCUS T homolog are involved in poor-nutrition stress-induced flowering of Pharbitis nil.

The short-day plants Pharbitis nil (synonym Ipomoea nil), var. Violet and Tendan were grown in a diluted nutrient solution or tap water for 20 days under long-day conditions. Violet plants were induced to flower and vegetative growth was inhibited, whereas Tendan plants were not induced to flower, although vegetative growth was inhibited under these conditions. The Violet plants induced to flower by poor-nutrition stress produced fertile seeds and their progeny developed normally. Defoliated Violet scions grafted onto the rootstocks of Violet or Tendan were induced to flower under poor-nutrition stress conditions, but Tendan scions grafted onto the Violet rootstocks were not induced to flower. These results indicate that a transmissible flowering stimulus is involved in the induction of flowering by poor-nutrition stress. The poor-nutrition stress-induced flowering was inhibited by aminooxyacetic acid, a phenylalanine ammonia-lyase inhibitor, and this inhibition was almost completely reversed by salicylic acid (SA). However, exogenously applied SA did not induce flowering under non-stress conditions, suggesting that SA may be necessary but not sufficient to induce flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the Violet plants were induced to flower by growing in tap water, but expression of PnFT1, another ortholog of FT, was not induced, suggesting the specific involvement of PnFT2 in stress-induced flowering.

Light- and IAA-regulated ACC synthase gene (PnACS) from Pharbitis nil and its possible role in IAA-mediated flower inhibition.

The light- and indole-3-acetic acid (IAA)-regulated 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (PnACS) from Pharbitis nil was isolated. Here, it was shown that the gene was expressed in cotyledons, petioles, hypocotyls, root and shoot apexes both in light- and dark-grown seedlings. The highest expression level of PnACS was found in the roots. IAA applied to the cotyledons of P. nil seedlings caused a clear increase of PnACS messenger accumulation in all the organs examined. In this case, the most IAA-responsive were the hypocotyls. Our studies revealed that the PnACS transcript level in the cotyledons exhibited diurnal oscillations under both long-day (LD) and short-day (SD) conditions. IAA applied at the beginning of inductive darkness caused a dramatic increase in the expression of PnACS, suggesting that the inhibitory effect of IAA on P. nil flowering may result from its stimulatory effect on ethylene production.

Structure and biological activity of novel FN analogs as flowering inducers.

(12Z,15Z)-9-Hydroxy-10-oxooctadeca-12,15-dienoic acid (1) and norepinephrine (2) undergo cycloaddition to afford FN1 (3) and FN2 (4), both of which induce flowering in Lemna paucicostata. Although the derivatives of 1 were suggested to also yield FN-like compounds after reacting with 2, their structures have not been elucidated. In this report, we investigated the structure and stereochemistry of seven novel FN analogs. These analogs were shown to be formed in the same regio- and stereocontrolled manner as FNs. Moreover, the activity of FN analogs on flowering induction was investigated, and we determined that all analogs, except for compound 8, were effective flowering inducers for L. paucicostata.

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.

The gravity-regulated growth of axillary buds is mediated by a mechanism different from decapitation-induced release.

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.

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.

Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation.

The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria, whose flowering state is stable and the short day plant Pharbitis nil, whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens. On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state.

A reappraisal of the role of abscisic acid and its interaction with auxin in apical dominance.

BACKGROUND AND AIMS: Evidence from pea rms1, Arabidopsis max4 and petunia dad1 mutant studies suggest an unidentified carotenoid-derived/plastid-produced branching inhibitor which moves acropetally from the roots to the shoots and interacts with auxin in the control of apical dominance. Since the plant hormone, abscisic acid (ABA), known to inhibit some growth processes, is also carotenoid derived/plastid produced, and because there has been indirect evidence for its involvement with branching, a re-examination of the role of ABA in apical dominance is timely. Even though it has been determined that ABA probably is not the second messenger for auxin in apical dominance and is not the above-mentioned unidentified branching inhibitor, the similarity of their derivation suggests possible relationships and/or interactions. METHODS: The classic Thimann-Skoog auxin replacement test for apical dominance with auxin [0.5 % naphthalene acetic acid (NAA)] applied both apically and basally was combined in similar treatments with 1 % ABA in Ipomoea nil (Japanese Morning Glory), Solanum lycopersicum (Better Boy tomato) and Helianthus annuus (Mammoth Grey-striped Sunflower). KEY RESULTS: Auxin, apically applied to the cut stem surface of decapitated shoots, strongly restored apical dominance in all three species, whereas the similar treatment with ABA did not. However, when ABA was applied basally, i.e. below the lateral bud of interest, there was a significant moderate repression of its outgrowth in Ipomoea and Solanum. There was also some additive repression when apical auxin and basal ABA treatments were combined in Ipomoea. CONCLUSION: The finding that basally applied ABA is able partially to restore apical dominance via acropetal transport up the shoot suggests possible interactions between ABA, auxin and the unidentified carotenoid-derived branching inhibitor that justify further investigation.

Inhibitory role of gibberellins in theobroxide-induced flowering of Pharbitis nil.

Theobroxide, a novel active compound isolated from a fungus, has been reported previously to induce potato tuberization and flower bud formation in Pharbitis nil under non-inductive long-day conditions. Up to date, the action mechanism of theobroxide on flower-bud induction of P. nil, however, is still unknown. In the present study, we observed a reduction of the stem length, along with the induction of flower buds, in theobroxide-treated and short-day-grown P. nil plants. Also, the results showed that flower bud formation was delayed markedly in P. nil seedlings with removal of cotyledons or exposure to night break. The suppression effect of night-break and cotyledon-removal, however, was abolished completely by spraying theobroxide. Endogenous gibberellin(1/3) contents in P. nil plants treated with theobroxide or grown under short-day conditions were relatively lower, suggesting that gibberellins probably are negatively involved in theobroxide- and short-day-induced flower-bud formation of P. nil.