Differential effects of light-to-dark transitions on phase setting in circadian expression among clock-controlled genes in Pharbitis nil.

The circadian clock is synchronized by the day-night cycle to allow plants to anticipate daily environmental changes and to recognize annual changes in day length enabling seasonal flowering. This clock system has been extensively studied in Arabidopsis thaliana and was found to be reset by the dark to light transition at dawn. By contrast, studies on photoperiodic flowering of Pharbitis nil revealed the presence of a clock system reset by the transition from light to dark at dusk to measure the duration of the night. However, a Pharbitis photosynthetic gene was also shown to be insensitive to this dusk transition and to be set by dawn. Thus Pharbitis appeared to have two clock systems, one set by dusk that controls photoperiodic flowering and a second controlling photosynthetic gene expression similar to that of Arabidopsis. Here, we show that circadian mRNA expression of Pharbitis homologs of a series of Arabidopsis clock or clock-controlled genes are insensitive to the dusk transition. These data further define the presence in Pharbitis of a clock system that is analogous to the Arabidopsis system, which co-exists and functions with the dusk-set system dedicated to the control of photoperiodic flowering.

Genomic structure and promoter characterization of the CDPK kinase gene expressed during seed formation in Pharbitis nil.

CDPK kinases are a unique class of calcium sensor/responders that regulate many growth and developmental processes as well as stress responses of plants. PnCDPK1 kinase from Pharbitis nil is regulated by light and contributes to seed germination, seedling growth and flower formation. Following an earlier work in which we identified the PnCDPK1 coding sequence and a 330bp long 3'UTR (untranslated region), we present for the first time the genomic organization of PnCDPK1, including intron analysis and the gene copy number designation. We completed the research by identifying the 5'-flanking region of PnCDPK1 and analyzed it in silico, which led to the discovery of several cis-regulatory elements involved in light regulation, embryogenesis and seed development. The functional analysis of P. nil CDPK showed characterization of the PnCDPK1 transcript and PnCDPK protein level during seed formation and fruit maturation. The greatest amount of PnCDPK1 mRNA was present in the last stages of seed maturation. Moreover, two PnCDPK proteins of different molecular masses were discovered during fruit development, showing various protein accumulation and activity profile. The 56kDa protein dominated in the early stages of fruit development, whereas the smaller protein (52kDa) was prominent in the latter stages.

Constitutive expression of the GIGANTEA ortholog affects circadian rhythms and suppresses one-shot induction of flowering in Pharbitis nil, a typical short-day plant.

GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function in Arabidopsis. Mutations in the GI gene cause photoperiod-insensitive flowering and altered circadian rhythms. We isolated the GI ortholog PnGI from Pharbitis (Ipomoea) nil, an absolute short-day (SD) plant. PnGI mRNA expression showed diurnal rhythms that peaked at dusk under SD and long-day (LD) conditions, and also showed robust circadian rhythms under continuous dark (DD) and continuous light (LL) conditions. Short irradiation with red light during the flower-inductive dark period did not change PnGI expression levels, suggesting that such a night break does not abolish flowering by affecting the expression of PnGI. In Pharbitis, although a single dusk signal is sufficient to induce expression of the ortholog of FLOWERING LOCUS T (PnFT1), PnGI mRNA expression was not reset by single lights-off signals. Constitutive expression of PnGI (PnGI-OX) in transgenic plants altered period length in leaf-movement rhythms under LL and affected circadian rhythms of PnFT mRNA expression under DD. PnGI-OX plants formed fewer flower buds than the wild type when one-shot darkness was given. In PnGI-OX plants, expression of PnFT1 was down-regulated, suggesting that PnGI functions as a suppressor of flowering, possibly in part through down-regulation of PnFT1.

Reduction in the critical dark length for flower induction during aging in the short-day plant Pharbitis nil var. Kidachi.

The stress-sensitive short-day plant Pharbitis nil var. Kidachi flowers under a 16-h light and 8-h dark regime and non-stress conditions when grown for long periods of time. Such flowering was found to occur from the third week, and the floral buds were formed from the eighth node of the main stem. When young plants were grafted onto aged plants, the scions were induced to flower early. This flower induction by grafting was more effective when older plants were used as rootstocks. Grafting experiments using a single leaf as a donor revealed that younger leaves are more responsive to flower induction, suggesting that this age-mediated flowering response is not induced by aging or senescence of individual leaves. Rather, the plant may obtain the ability to flower as the whole plant ages. Flowering does not occur under continuous light conditions. A night break given in the 8-h dark period inhibits flowering. These results suggest that 8-h dark conditions, which are normally considered to be long-day conditions, actually correspond to short-day conditions for this plant. The 8-h dark conditions caused early flowering more efficiently in older plants. The critical dark length determined by a single treatment was 12 h in 0-week-old plants and was reduced to 6 h in 2- and 4-week-old plants. These results suggest that the critical dark length becomes shorter when plants get older. The expression of PnFT1 and PnFT2, orthologs of the flowering gene flowering locus T, was analyzed by reverse transcription-polymerase chain reaction revealing that the expression of PnFT at the end of dark period is correlated with 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.

Effect of light on soluble guanylyl cyclase activity in Pharbitis nil seedlings.

Cyclic GMP acts as a chemical switch in plant cells to modulate cellular reactions. However, its metabolism has not been extensively explored and is still poorly understood. Previous experiments suggest that an endogenous cGMP system could participate in the mechanism of phytochrome controlled photoperiodic flower induction in Pharbitis nil. In order to gain further information on the role of cGMP, we have begun to study the enzyme of cGMP synthesis. In this article, the presence of the enzyme with guanylyl cyclase (GC) activity in soluble protein fractions of P. nil is reported. A large portion of the enzymatic activity is present in the cotyledons, where enzyme activity amounted to 0.45 pmol cGMP/min/mg protein. The enzyme exhibited a K(m) 0.5mM for GTP. A plot of 1/v versus 1/[GTP] was linear and V(max) was 0.74 pmol cGMP/min/mg protein. It was shown that the anti-sGC antibody recognise a 40 kDa protein. Moreover, the NO-donor, sodium nitroprusside (SNP) and YC-1, as a NO-independent stimulator, enhanced enzyme activity. The NS 2028 (a potent GC inhibitor) treatments provoked a 3-fold reduction of the enzyme activity in comparison to the untreated fractions. Furthermore, the influence of light on GC activity was analysed. It was noted that cGMP level increased in cool white light, and darkness inhibited enzyme activity. Exposure to blue light acts to stimulate cGMP formation, whereas in red light a rapid decrease in GC activity was observed that returned to the high level when far-red light was applied after the red light treatment. The results presented in this work strongly argue that an enzyme with guanylyl cyclase activity is present in P. nil organs and its activity is controlled by light via the photoreceptors-dependent pathways.

Intracellular distribution of phototropin 1 protein in the short-day plant Ipomoea nil.

Phototropin 1 (phot1) is a blue-light Ser/Thr receptor kinase that contains two LOV domains. It is a plasma membrane-associated protein that mediates phototropism, blue-light induced chloroplast movement, and stomatal opening. The aim of the present work was to analyze the intracellular localization of phot1 protein in Ipomoea nil seedlings. In cotyledon and hypocotyl cells of etiolated seedlings, phot1 was specifically localized in the plasma membrane regions, whereas in light-treated seedlings, it was homogeneously distributed throughout the whole cytoplasm, excluding cell nuclei and vacuoles. Phot1 was also localized in cotyledon epidermal and guard cells. Such a localization pattern suggests a light-dependent intracellular distribution of phot1 in Ipomoea nil. On the basis of the spatial distribution, the possible role of phot1 is also discussed.

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.

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.