Photoperiodic Regulation Of Flowering Research Articles

Comparative Genomics and Functional Characterisation of the GIGANTEA Gene from the Temperate Forage Perennial Ryegrass Lolium perenne

Lolium perenne (ryegrass) is a perennial forage grass of worldwide importance. It is a temperate climate grass and flowering is induced by long day photoperiods. The agronomic productivity of ryegrass is strongly influenced by flowering time, but less is known about ryegrass flowering time regulators in other temperate Poaceae like wheat, barley and the model grass Brachypodium. The GIGANTEA (GI) gene was first identified in Arabidopsis and is an important regulator of photoperiodic flowering in angiosperms. GI usually exists as a conserved, single-copy gene. However, recently, genome sequencing has revealed that some plants, including the tropical grass maize, carry more than one full-length copy of GI. Here we describe the isolation and characterisation of the ryegrass L. perenne GIGANTEA gene (LpGI) gene. Comparative genomic analysis indicates that LpGI gene structure is very well conserved overall with GI genes from monocots and eudicots. LpGI protein clusters with GI proteins from other temperate grasses and is most closely related to the GI protein from meadow fescue. Genetic mapping locates LpGI to a chromosomal region that is syntenic with rice and Brachypodium GI. Our functional characterisation shows that LpGI shows a diurnal pattern of expression, continues to oscillate under constant light and adjusts its phase in response to changes in day length as seen in other plants. Constitutive expression of LpGI completely rescues the Arabidopsis gigantea-3 allele (gi-3) mutation, confirming that the isolated ryegrass gene is fully functional. Taken together, it is highly likely that LpGI is orthologous to Arabidopsis GI and involved in photoperiodic flowering time control in ryegrass.

Mapping-by-sequencing identifies HvPHYTOCHROME C as a candidate gene for the early maturity 5 locus modulating the circadian clock and photoperiodic flowering in barley.

Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering.

GmFT2a and GmFT5a redundantly and differentially regulate flowering through interaction with and upregulation of the bZIP transcription factor GmFDL19 in soybean.

FLOWERING LOCUS T (FT) is the key flowering integrator in Arabidopsis (Arabidopsis thaliana), and its homologs encode florigens in many plant species regardless of their photoperiodic response. Two FT homologs, GmFT2a and GmFT5a, are involved in photoperiod-regulated flowering and coordinately control flowering in soybean. However, the molecular and genetic understanding of the roles played by GmFT2a and GmFT5a in photoperiod-regulated flowering in soybean is very limited. In this study, we demonstrated that GmFT2a and GmFT5a were able to promote early flowering in soybean by overexpressing these two genes in the soybean cultivar Williams 82 under noninductive long-day (LD) conditions. The soybean homologs of several floral identity genes, such as GmAP1, GmSOC1 and GmLFY, were significantly upregulated by GmFT2a and GmFT5a in a redundant and differential pattern. A bZIP transcription factor, GmFDL19, was identified as interacting with both GmFT2a and GmFT5a, and this interaction was confirmed by yeast two-hybridization and bimolecular fluorescence complementation (BiFC). The overexpression of GmFDL19 in soybean caused early flowering, and the transcription levels of the flowering identity genes were also upregulated by GmFDL19, as was consistent with the upregulation of GmFT2a and GmFT5a. The transcription of GmFDL19 was also induced by GmFT2a. The results of the electrophoretic mobility shift assay (EMSA) indicated that GmFDL19 was able to bind with the cis-elements in the promoter of GmAP1a. Taken together, our results suggest that GmFT2a and GmFT5a redundantly and differentially control photoperiod-regulated flowering in soybean through both physical interaction with and transcriptional upregulation of the bZIP transcription factor GmFDL19, thereby inducing the expression of floral identity genes.

The BBX family of plant transcription factors

The B-box (BBX) proteins are a class of zinc-finger transcription factors containing a B-box domain with one or two B-box motifs, and sometimes also feature a CCT (CONSTANS, CO-like, and TOC1) domain. BBX proteins are key factors in regulatory networks controlling growth and developmental processes that include seedling photomorphogenesis, photoperiodic regulation of flowering, shade avoidance, and responses to biotic and abiotic stresses. In this review we discuss the functions of BBX proteins and the role of B-box motif in mediating transcriptional regulation and protein-protein interaction in plant signaling. In addition, we provide novel insights into the molecular mechanisms of their action and the evolutionary significance of their functional divergence.

Small RNAs and the Big Decisions: MicroRNA Regulation of Photoperiodic Flowering in Brachypodium distachyon

Should I make flowers now, or wait and maybe develop a few more leaves? It’s a decision with important implications for reproductive success of wild plants and yields of domesticated plants. Plants can't call a friend to ask for advice on this big decision; instead, they rely on their ability to

Solar rhythm in the regulation of photoperiodic flowering of long-day and short-day plants

In photoperiodic flowering, long-day (LD) plants are induced to flower seasonally when the daylight hours are long, whereas flowering in short-day (SD) plants is promoted under short photoperiods. According to the widely accepted external coincidence model, flowering occurs in LD Arabidopsis when the circadian rhythm of the gene CONSTANS (CO) peaks in the afternoon, when it is light during long days but dark when the days are short. Nevertheless, extending this explanation to SD flowering in rice, Oriza sativa, requires LD and SD plants to have 'opposite light requirements' as the CO orthologue in rice, HEADING-DATE1 (Hd1), promotes flowering only under short photoperiods. This report proposes a role of the plant's solar rhythm in promoting seasonal flowering. The interaction between rhythmic genes entrained to the solar clock and those entrained to the circadian clock form the basis of an internal coincidence model that explains both LD and SD flowering equally well. The model invokes no presumption of opposite light requirements between LD and SD plants, and further argues against any specific requirement of either light or darkness for SD flowering. Internal coincidence predicts the inhibition of SD flowering of the rice plant by a night break (a brief interruption of light), while it also provides a plausible explanation for how a judiciously timed night break promotes Arabidopsis flowering even on short days. It is the timing of the light transitions (sunrise and sunset) rather than the duration of light or darkness per se that regulates photoperiod-controlled flowering.

Ubiquitin-Specific Proteases UBP12 and UBP13 Act in Circadian Clock and Photoperiodic Flowering Regulation in Arabidopsis

Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.

ELF4 Regulates GIGANTEA Chromatin Access through Subnuclear Sequestration

Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI), one of the key clock components in Arabidopsis, makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4) as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS (CO), to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering.

Ehd4 Encodes a Novel and Oryza-Genus-Specific Regulator of Photoperiodic Flowering in Rice

Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the “florigen” genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.

OsELF3-1, an Ortholog of Arabidopsis EARLY FLOWERING 3, Regulates Rice Circadian Rhythm and Photoperiodic Flowering

Arabidopsis thaliana EARLY FLOWERING 3 (ELF3) as a zeitnehmer (time taker) is responsible for generation of circadian rhythm and regulation of photoperiodic flowering. There are two orthologs (OsELF3-1 and OsELF3-2) of ELF3 in rice (Oryza sativa), but their roles have not yet been fully identified. Here, we performed a functional characterization of OsELF3-1 and revealed it plays a more predominant role than OsELF3-2 in rice heading. Our results suggest OsELF3-1 can affect rice circadian systems via positive regulation of OsLHY expression and negative regulation of OsPRR1, OsPRR37, OsPRR73 and OsPRR95 expression. In addition, OsELF3-1 is involved in blue light signaling by activating EARLY HEADING DATE 1 (Ehd1) expression to promote rice flowering under short-day (SD) conditions. Moreover, OsELF3-1 suppresses a flowering repressor GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) to indirectly accelerate flowering under long-day (LD) conditions. Taken together, our results indicate OsELF3-1 is essential for circadian regulation and photoperiodic flowering in rice.

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.

The MedicagoFLOWERING LOCUS THomolog,MtFTa1, Is a Key Regulator of Flowering Time

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.

Characterization and transgenic study of CONSTANS-LIKE8 (COL8) gene in Arabidopsis thaliana: expression of 35S:COL8 delays flowering under long-day conditions

CONSTANS (CO) is a regulator of photoperiodic flowering that activates the transcription of a florigen gene, FLOWERING LOCUS T (FT), in Arabidopsis. CO belongs to the CO/CONSTANS-LIKE (COL) protein family, which consists of 17 proteins and is subclassified into three groups. Here, we characterized the gene for one of the group II members, COL8. The COL8 mRNA accumulated in seeds, leaves, flowers, and siliques. Transgenic Arabidopsis plants with a beta-glucuronidase (GUS) reporter gene driven by a 3-kb COL8 promoter displayed strong GUS activity in leaves. Both transiently and stably produced fluorescence-tagged COL8 proteins were localized in the nucleus. Transgenic Arabidopsis plants possessing COL8 cDNA driven by a cauliflower mosaic virus (CaMV) 35S promoter did not show any altered circadian rhythm under constant light conditions, but showed a late-flowering phenotype under long-day conditions. In these transgenic plants, CO mRNA did not decrease from zeitgeber time (zt) 12 to zt 16, whereas FT mRNA decreased from zt 8 to zt 24. The possible mechanisms for the late-flowering phenotype of the COL8 plants driven by the CaMV 35S promoter are discussed.

EFO1 and EFO2, encoding putative WD-domain proteins, have overlapping and distinct roles in the regulation of vegetative development and flowering of Arabidopsis

From screening a population of Arabidopsis overexpression lines, two Arabidopsis genes were identified, EFO1 (early flowering by overexpression 1) and EFO2, that confer early flowering when overexpressed. The two genes encode putative WD-domain proteins which share high sequence similarity and constitute a small subfamily. Interestingly, the efo2-1 loss-of-function mutant also flowered earlier in short days and slightly earlier in long days than the wild type, while no flowering-time or morphological differences were observed in efo1-1 relative to the wild type. In addition, the efo2-1 mutation perturbed hypocotyl elongation, leaf expansion and formation, and stem elongation. EFO1 and EFO2 are both regulated by the circadian clock. Expression and genetic analyses revealed that EFO2 suppresses flowering largely through the action of CONSTANS (CO) and flowering locus T (FT), suggesting that EFO2 is a negative regulator of photoperiodic flowering. The growth defects in efo2-1 were augmented in efo1 efo2, but the induction of FT in the double mutant was comparable to that in efo2-1. Thus, while EFO2 acts as a floral repressor, EFO1 may not be directly involved in flowering, but the two genes do have overlapping roles in regulating other developmental processes. EFO1 and EFO2 may function collectively to serve as one of the converging points where the signals of growth and flowering intersect.

Expression, purification and characterisation of GIGANTEA: A circadian clock-controlled regulator of photoperiodic flowering in plants

The Arabidopsis thaliana (Arabidopsis) GIGANTEA (GI) gene is a central component of the photoperiodic flowering pathway. While it has been 40 years since the first mutant alleles of GI were described much is still unknown about the molecular mechanism of GI action. To investigate the biochemistry and domain organisation (and ultimately to give a greater understanding of the role of GI in floral induction), it is first necessary to produce significant quantities of purified protein. Soluble affinity-tagged full-length GI was expressed in Escherichia coli (E. coli) and was stabilised by the addition of the detergent n-dodecyl-β-d-maltoside (DDM) to storage and purification buffers. Stabilised GI was purified using a variety of chromatographic methods, and characterised using a selection of biochemical techniques including circular dichroism, and dynamic light scattering. This showed that purified GI contained secondary structure, but was polydisperse in solution. Electron microscopy suggests a possible tetramer arrangement of GI. Limited proteolytic digests and mass spectrometry were used to identify potential GI domains. This led to the identification of a predicted 46kDa amino-terminal GI domain. GI was also expressed in Sf9 insect cells using the baculovirus expression system. GI produced via this route gave insoluble protein.

Evolutionarily conserved photoperiod mechanisms in plants

Day-length and the circadian clock control critical aspects of plant development such as the onset of reproduction by the photoperiodic pathway.1 CONSTANS (CO) regulates the expression of a florigenic mobile signal from leaves to the apical meristem and thus is central to the regulation of photoperiodic flowering.2 This regulatory control is present in all higher plants,3 but the time in evolution when it arose was unknown. We have shown that the genomes of green microalgae encode members of the CONSTANS-like (COL) protein family. One of these genes, the Chlamydomonas reinhardtii CO homolog (CrCO), can complement the co mutation in Arabidopsis.4 CrCO expression is controlled by the clock and photoperiod in Chlamydomonas and at the same time is involved in the correct timing of several circadian output processes such as the accumulation of starch or the coordination of cell growth and division. We have proposed that, since very early in the evolutionary lineage that gave rise to higher plants, CO homologs have been involved in the photoperiod control of important developmental processes, and that the recruitment of COL proteins in other roles may have been crucial for their evolutionary success.

Association of the circadian rhythmic expression of GmCRY1a with a latitudinal cline in photoperiodic flowering of soybean

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor CRY2 regulates photoperiodic flowering in the experimental model plant Arabidopsis thaliana. However, it is unclear whether genetic variations affecting cryptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two cryptochromes in soybean, GmCRY1a and GmCRY2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) exhibit a strong latitudinal cline in photoperiodic flowering. Similar to their Arabidopsis counterparts, both GmCRY1a and GmCRY2a affected blue light inhibition of cell elongation, but only GmCRY2a underwent blue light- and 26S proteasome-dependent degradation. However, in contrast to Arabidopsis cryptochromes, soybean GmCRY1a, but not GmCRY2a, exhibited a strong activity promoting floral initiation, and the level of protein expression of GmCRY1a, but not GmCRY2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmCRY1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmCRY1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmCRY1a protein play an important role in determining latitudinal distribution of soybeans.

CONSTANS delays flowering and affects tuber yield in potato

CONSTANS (CO) has a central role in the photoperiodic regulation of flowering in Arabidopsis thaliana. We show here that potato (Solanum tuberosum ssp. andigena) plants constitutively expressing Arabidopsis CO (pACo plants) flower late under all photoperiodic conditions tested. Exogenous application of gibberellic acid to pACo plants corrected their short stem phenotype but not their late flowering. To further understand the effect of CO in potato, we used three photoperiodic conditions: short days (SD), which strongly induce tuberisation of wild type plants, SD supplemented with a night break (SD+NB), which are moderately inductive, and tuberisation-inhibiting long days. Tuberisation of pACo plants was delayed under SD and very strongly delayed or completely inhibited under SD+NB, suggesting that CO affects an autonomous pathway controlling potato tuberisation. In addition, tuber yield, a trait of high agronomic relevance, was significantly increased in pACo plants expressing moderate CO levels. Our results indicate that CO affects flowering and stem elongation through distinct mechanisms and suggest that its effects on flowering and tuberisation in potato are photoperiod-independent.

Metabolism of α-ketol derivative of linolenic acid (KODA), a flowering-related compound, in Pharbitis nil

α-Ketol of octadecadienoic acid (KODA, 1) has been suggested to play a role in the photoperiod-regulated flowering in Pharbitis nil. The level of 1 in cotyledons is temporarily controlled during short-day conditions. The biosynthesis of 1 is well studied in plants; however, its in vivo conversion is less understood. We have investigated this issue by studying the metabolism of exogenously-applied [U- 13C]- 1, [1- 14C]- 1, and non-labeled 1 in P. nil seedlings by the spectroscopic methods and identified six major metabolites ( 4– 9). We have also found that the enantiomers of 1 are differentially metabolized in P. nil seedlings.

A Putative CCAAT-Binding Transcription Factor Is a Regulator of Flowering Timing in Arabidopsis

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.