Epigenetic regulation of photoperiodic flowering

The cytidine analogue 5-azacytidine, which causes DNA demethylation, induced flowering in the non-vernalization-requiring plants Perilla frutescens var. crispa, Silene armeria and Pharbitis nil (synonym Ipomoea nil) under non-inductive photoperiodic conditions, suggesting that the expression of photoperiodic flowering-related genes is regulated epigenetically by DNA methylation. The flowering state induced by DNA demethylation was not heritable. Changes in the genome-wide methylation state were examined by methylation-sensitive amplified fragment length polymorphism analysis. This analysis indicated that the DNA methylation state was altered by the photoperiodic condition. DNA demethylation also induced dwarfism, and the induced dwarfism of P. frutescens was heritable.Key words: 5-azacytidine, DNA methylation, photoperiodic flowering, epigenetics, methylation-sensitive amplified fragment length polymorphism, CpG island, dwarfism     

Induction of flowering in the duckweed Lemna paucicostata under non-inductive photoperiods by tannic acid

Flowering in Lemna paucicostata 6746 could be induced by tannic acid under strictly non-inductive photoperiods. This polyphenol completely abolished the photoperiodic sensitivity of strain 6746 as flowering could also be obtained under continuous light (nearly 80% flowering was recorded in the plants supplied with 10⁻⁵ tannic acid). Though its mode of action is unknown, tannic acid is unlikely to act as a gibberellin-antagonist in its effect on flowering in strain 6746.     

Substances of plant flowering

The investigation of the hormonal nature of plant flowering in connection with their photoperiodic reaction has shown that flowering depends on a bicomponental system of hormones, gibberellins regulating stem formation and growth and substances of the anthesin type regulating flower formation. In agreement with the division of the photoperiodic reaction into a leaf and a stem phase the study of the internal factors acting on plant flowering was carried out by means of leaf and stem (apex, bud and callus) models. The results obtained from work with leaf models proved the presence of two groups of hormones of flowering in plants. The data obtained from the application of stem models pointed to the localization of the action of gibberellin and anthesin in different zones of the shoot apices and characterized the potential capacity for flower formation of isolated callus tissue of neutral and photoperiodically sensitive species.     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Regulation of flowering plants of different biotypes

The patterns of control of flowering are analyzed in plants of different biotypes. The photoperiodic reaction of flowering taken as an example, the whole net of control is considered: from the environmental stimulus through its physiological transformation in the leaf in the corresponding hormonal impulse which, in turn, controls the realization of genetic programme and formation of generative organs in the stem buds. The photoperiodically neutral plants taken as an example, the patterns of age control of flowering are considered. In plants of different photoperiodic groups the synthesis of complementary components of florigen was shown to proceed either autonomously under the photoperiodic effect or under the inducing effect of definite photoperiods. The autonomous and inducible mechanisms of biosynthesis of the flowering hormones have a common base, the genetic system to which the environment sends its stimuli through the hormonal interactions. The interaction of hormonal and genetic developmental factors is considered, the evocation of flowering in the stem buds taken as an example.     

Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method

To better understand the molecular mechanisms of the photoperiodic regulation of rice, a short-day plant, we isolated 27 cDNAs that were differentially expressed in the photoperiod-insensitive se5 mutant from approximately 8,400 independent mRNA species by the use of a fluorescent differential display (FDD). For this screening, we isolated mRNAs at five different time points during the night and compared their expression patterns between se5 and the wild type. Of 27 cDNAs isolated, 12 showed diurnal expression patterns often associated with genes involved in the determination of the flowering time. In se5, expression of nine cDNAs was increased. Five of these cDNAs were up-regulated under SD, suggesting that they may promote flowering under SD. They included genes encoding a cDNA containing a putative NAC domain, the fructose-bisphosphate aldolase, and a protease inhibitor. Expression of three cDNAs was decreased in se5 but not photoperiodically regulated. These cDNAs included a rice homolog of Arabidopsis GIGANTEA (GI), lir1, and a gene for myo-inositol 1-phosphate synthase, all of which were previously shown to be under the control of circadian clocks. The expression patterns of the rice homolog of GI, OsGI, were similar to those of the Arabidopsis GI, suggesting the conservation of some mechanisms for the photoperiodic regulation of flowering between these two species.     

Role of VIN3-LIKE 2 in facultative photoperiodic flowering response in Arabidopsis

In Arabidopsis, expression of FLC and FLC-related genes (collectively called FLC clade) contributes to flowering time in response to environmental changes, such as day length and temperature, by acting as floral repressors. VIN3 is required for vernalization-mediated FLC repression and a VIN3 related protein, VIN3-LIKE 1/VERNALIZATION 5 (VIL1/VRN5), acts to regulate FLC and FLM in response to vernalization.^1–3 VIN3 also exists as a small family of PHD finger proteins in Arabidopsis, including VIL1/VRN5, VIL2/VEL1, VIL3/VEL2 and VIL4/VEL3. We showed that the PHD finger protein, VIL2, is required for proper repression of MAF5, an FLC clade member, to accelerate flowering under non-inductive photoperiods. VIL2 acts together with POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) to repress MAF5 in a photoperiod dependent manner.Key words: photoperiod, chromatin, floweringThe decision to flower is critical to the survival of flowering plants. Thus, plants sense environmental cues to initiate floral transition at a time that both ensures and optimizes their own reproductive fitness. Using a model plant, Arabidopsis thaliana, genetic studies have shown that the regulation of floral transition mainly consists of four genetic pathways: the inductive photoperiod pathway, the autonomous pathway, the vernalization pathway and the gibberellin pathway.⁴ In Arabidopsis, these four flowering pathways eventually merge into a group of genes called floral integrators, including FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and LEAFY (LFY). Based on the response to specific photoperiod conditions, the flowering behaviors of plants can be classified into three groups: long day (LD), short day (SD) and day neutral response.^5,6 Depending on the requirement of day length, plants show either obligate or facultative responses. For example, henbane, carnation and ryegrass are obligate long day (LD) flowering plants which flower under increasing inductive photoperiod but do not flower at all under non-inductive photoperiod.⁵ On the other hand, plants including Arabidopsis, wheat, lettuce and barley, are considered to be facultative flowering plants. Thus, these plants exhibit early flowering under LD and late-flowering under non-inductive short days (SD). Studies on photoperiodic flowering time mainly focus on the inductive LD-photoperiod pathway in Arabidopsis.     

Flowering induced by 5-azacytidine, a DNA demethylating reagent in a short-day plant, Perilla frutescens var. crispa

Treatment with 5-azacytidine, a DNA demethylating reagent, induced flowering in Perilla frutescens (L.) Britton var. crispa (Thunb. ex Murray) Decne. ex L. H. Bailey, an absolute short-day plant under long days. The 5-azacytidine treatment induced slight suppression of vegetative growth but had no obvious effect on any other phenotypes. The Southern hybridization analysis of the genomic DNA isolated from the leaves of 5-azacytidine-treated plants and digested with restriction enzyme, methylation-insensitive Msp I or methylation-sensitive Hpa II with P. frutescens 25S-18S rDNA intergenic spacer probe indicated that the 5-azacytidine treatment caused demethylation of the genomic DNA. The 5-azacytidine-induced flowering was delayed as compared with the short day-induced flowering. Flowers were formed even at the lower nodes which had not been directly treated with 5-azacytidine. The results suggest that DNA demethylation induced flowering by inducing the production of a transmissible flowering stimulus in P. frutescens .     

The role of light quality of photoperiodic lighting on photosynthesis,flowering and metabolic profiling in Ranunculus asiaticus L.

Photoperiodic light quality affects flowering of long day plants, by influencing the phytochrome photoequilibria (PPE) at plant level; however, the most effective light spectrum to promote flowering is still unknown for most of the flower crops. We evaluated the influence of light spectrum of three light sources, with different induced PPE, on photosynthesis, metabolic profiling, plant growth and flowering in two hybrids of Ranunculus asiaticus L., MBO (early flowering) and MDR (medium earliness). Three photoperiodic treatments were compared to natural day length (NL): white fluorescent light (PPE 0.84), light emitting diodes (LEDs) with red:far red (R:FR) light at 3:1 ratio (PPE, 0.84) and LEDs with R:FR light at 1:3 ratio (PPE 0.63). Under natural light, net photosynthesis was higher in MDR than in MBO, while photochemistry was similar in the hybrids. Compared to NL, photoperiodic treatments did not affect net photosynthesis, while they promoted the quantum yield of PSII and reduced the non-photochemical quenching. Under NL, plant growth was greater in MBO, while flowering started earlier in MDR and flowers characteristics were similar in the hybrids. Despite the greater sensitivity of MDR plants in terms of metabolism, photoperiodic lighting improved plant growth and reduced the flowering time only in MBO, with a stronger effect under R:FR 3:1 light. MDR plants were characterized by higher soluble sugars, polyphenols, photosynthetic pigments and proteins, while MBO plants by higher starch and amino acid content. The morphological effects of photoperiodic light quality and the hybrid-specific response should be taken into account to optimize lighting protocols in commercial farms.     

CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums

Chrysanthemum is a typical short-day (SD) plant that responds to shortening daylength during the transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT)/Heading date 3a (Hd3a) plays a pivotal role in the induction of phase transition and is proposed to encode a florigen. Three FT-like genes were isolated from Chrysanthemum seticuspe (Maxim.) Hand.-Mazz. f. boreale (Makino) H. Ohashi & Yonek, a wild diploid chrysanthemum: CsFTL1, CsFTL2, and CsFTL3. The organ-specific expression patterns of the three genes were similar: they were all expressed mainly in the leaves. However, their response to daylength differed in that under SD (floral-inductive) conditions, the expression of CsFTL1 and CsFTL2 was down-regulated, whereas that of CsFTL3 was up-regulated. CsFTL3 had the potential to induce early flowering since its overexpression in chrysanthemum could induce flowering under non-inductive conditions. CsFTL3-dependent graft-transmissible signals partially substituted for SD stimuli in chrysanthemum. The CsFTL3 expression levels in the two C. seticuspe accessions that differed in their critical daylengths for flowering closely coincided with the flowering response. The CsFTL3 expression levels in the leaves were higher under floral-inductive photoperiods than under non-inductive conditions in both the accessions, with the induction of floral integrator and/or floral meristem identity genes occurring in the shoot apexes. Taken together, these results indicate that the gene product of CsFTL3 is a key regulator of photoperiodic flowering in chrysanthemums.     

Effect of varying lengths of inductive and supplementary non-inductive photoperiods on vegetative growth and flowering of Impatiens balsamina

The photoperiodic requirement for flowering in Impatiens balsaminachanges with the length of the photoperiod. Floral buds wereinitiated with two 8 hr but with four 15 hr photoperiods andflowers opened with four 8 hr but twenty-eight 15 hr photoperiods.A part of the photoperiodic requirement for floral inductionin this plant can be substituted by LDs containing 4 or morehours of darkness (10). It indicates the identical nature ofthe floral stimulus produced during the dark period, whetherit forms a part of the inductive or non-inductive cycles. Theeffect of these supplementary non-inductive photoperiodic cyclesin causing floral bud initiation also depends on the lengthof the first inductive obligatory cycle. More floral buds andflowers were produced on plants exposed to 15 hr than 8 hr photoperiods,probably due to the higher number of leaves that were producedunder the former condition of weaker induction. The shorterthe dark period in the photoperiodic cycle, the weaker the induction,the slower the rate of extension growth but the more differentiationof leaves. ¹ Present address: Department of Biology, Guru Nanak Dev University,Amritsar-143005, India. (Received November 9, 1977; )     

Bi-directional inflorescence development inArabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of paraclades

In this study we investigated Arabidopsis thaliana (L.) Heynh. inflorescence development by characterizing morphological changes at the shoot apex during the transition to flowering. Sixteen-hour photoperiods were used to synchronously induce flowering in vegetative plants grown for 30 d in non-inductive 8-h photoperiods. During the first inductive cycle, the shoot apical meristem ceased producing leaf primordia and began to produce flower primordia. The differentiation of paraclades (axillary flowering shoots), however, did not occur until after the initiation of multiple flower primordia from the shoot apical meristem. Paraclades were produced by the basipetal activation of buds from the axils of leaf primordia which had been initiated prior to photoperiodic induction. Concurrent with the activation of paraclades was the partial suppression of paraclade-associated leaf primordia, which became bract leaves. The suppression of bract-leaf primordia and the abrupt initiation of flower primordia during the first inductive photoperiod is indicative of a single phase change during the transition to flowering in photoperiodically induced Arabidopsis. Morphogenetic changes characteristic of the transition to flowering in plants grown continuously in 16-h photoperiods were qualitatively equivalent to the changes observed in plants which were photoperiodically induced after 30 d. These results suggest that Arabidopsis has only two phases of development, a vegetative phase and a reproductive phase; and that the production of flower primordia, the differentiation of paraclades from the axils of pre-existing leaf primordia and the elongation of internodes all occur during the reproductive phase.     

5-Fluorodeoxyuridine inhibition of photoperiodically induced flowering inChenopodium rubrum L.

Flowering in the short day plantChenopodium rubrum was inhibited by 5-fluoro-deoxyuridine (FDU) at a concentration of 4×10^?6 M and higher when applied during photoperiodic induction or immediately afterwards. This inhibition is always accompanied by a general reduction of growth (e.g. a decrease in the first leaf length). The mitotic activity within the shoot apex is completely blocked by FDU application during the photoperiodic treatment. The floral induction (evocationsensu Evans) was not cancelled in this situation as was revealed when reversing the FDU effect by thymidine application. One day after the end of the photoperiodic treatment (the plants were transferred to continuous light again) the FDU inhibition becomes irreparable. The results indicate that DNA synthesis and hence the mitotic activity are not obligatory prerequisites for photoperiodic floral induction inChenopodium. Low concentrations of FDU may promote flowering under suboptimal floral induction.     

The effects of hormones and saccharides on growth and flowering of green and herbicides-treatedChenopodium rubrum L. plants

The medium forin vitro culture of green and SANDOZ herbicides-treatedChenopodium rubrum L. plants contained saccharides and hormones in different concentrations. Five days after sowing, the plants were exposed to non-inductive (15 long days—LD) or inductive (6 short days—SD + 9 LD) photoperiodic conditions. The length of hypocotyl and cotyledon blade were measured and percentage of flowering was scored. Gibberellic acid (GA₃) stimulated hypocotyl growth of green and photobleached plants under SD and inhibited under LD conditions. Indole-3-acetic acid (IAA) slightly stimulated hypocotyl growth of green plants only under LD conditions. Benzylaminopurine (BAP) inhibited hypocotyl growth regardless of photoperiodic regime. The optimal concentration of glucose or saccharose for flowering in green and SANDOZ-treated plants was 5%. In green SAN 9785-treated plants exogenous saccharides compensated lack of photosynthates to bring about full flowering, but SAN 9789-treated plants needed in addition GA₃.     

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.     

Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant)

The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.     

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.     

The Effects of Photoperiod, Glucose and Gibberellic Acid on Growth In Vitro and Flowering of Chenopodium Murale

In vitro culture of long-day plant Chenopodium murale L was established. The effects of photoperiod, glucose and gibberellic acid (GA₃) on flowering and growth in vitro were investigated. Oscillatory changes of photoperiodic sensitivity were noticeable with regard to plant age. The plants induced at the phase of the 1^st and the 3^rd pair of leaves flowered to higher degree than those induced at the phase of 2^nd pair. Plants induced at the phase of the 1^st pair of leaves flowered to 17 % on 5 % glucose-containing medium and the addition of 5 mg dm^-3 GA₃ resulted in maximum flowering (43 %). Neither glucose nor GA₃ were able to compensate for photoperiodic requirements for flowering. Hypocotyl growth was decreased and the 1^st internode elongation and development of leaves were increased due to inductive photoperiodic conditions, as compared to non-inductive ones.     

Phloem transport of flowering signals

Seasonal variability in environmental parameters such as day length regulates many aspects of plant development. The transition from vegetative growth to flowering in Arabidopsis is regulated by seasonal changes in day length through a genetically defined molecular cascade known as the photoperiod pathway. Recent advances were made in understanding the tissues in which different components of the photoperiod pathway act to regulate floral induction. These studies highlighted the key role of the FT protein, which is produced in the leaves in response to inductive day lengths and traffics through the phloem to initiate flowering at the shoot apex. Unveiling the cellular and molecular details of this systemic signaling process will be required for a complete understanding of flowering regulation and other photoperiodic processes.