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Evolution of flowering in response to day length: flipping the CONSTANS switch.

Day length provides an important environmental cue by signalling conditions favourable for flowering. While Arabidopsis promotes flowering in response to long days, rice promotes flowering in response to short days. Despite this difference, a recent paper reveals that the network controlling this response is highly conserved in these distantly related plants, only the activity of one component is reversed. This reveals how an important developmental process can be diversified for adaptation by using the same set of genes, but regulating them differently.

Arabidopsis↗

Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability.

The four-member SPA protein family of Arabidopsis acts in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Here, we demonstrate that SPA proteins are, moreover, essential for photoperiodic flowering. Mutations in SPA1 cause phyA-independent early flowering under short day (SD) but not long day (LD) conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These spa1 spa3 spa4 triple mutants flower at the same time in LD and SD, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive SD. Among the four SPA genes, SPA1 is necessary and sufficient for normal photoperiodic flowering. Early flowering of SD-grown spa mutant correlates with strongly increased FT transcript levels, whereas CO transcript levels are not altered. Epistasis analysis demonstrates that both early flowering and FT induction in spa1 mutants is fully dependent on CO. Consistent with this finding, SPA proteins interact physically with CO in vitro and in vivo, suggesting that SPA proteins regulate CO protein function. Domain mapping shows that the SPA1-CO interaction requires the CCT-domain of CO, but is independent of the B-box type Zn fingers of CO. We further show that spa1 spa3 spa4 mutants exhibit strongly increased CO protein levels, which are not caused by a change in CO gene expression. Taken together, our results suggest, that SPA proteins regulate photoperiodic flowering by controlling the stability of the floral inducer CO.

Arabidopsis↗

[New alkylated derivates and dissociation constans of 3-hydroxy-1,2-benzisoxazole].

In the reactions of 3-hydroxy-1,2-benzisoxazole (BIO) and its 5-Cl and 6-Cl derivatives with ethyl bromoacetate were obtained the mixtures of O- and N- substituted esters but 7-NO2-BIO formed only O-ester. The products of N-aminomethylation of 5-NO2-BIO were active against Trichomonas vaginalis. The structures of pure compounds have been confirmed by elementary analysis and IR spectrometry. Determinations of dissociation constants (pKa) of BIO, 5-Cl-BIO, 6-Cl-BIO, 7-Cl-BIO, 5-F-BIO, 5-NO2-BIO, 7-NO2-BIO by potentiometric titration method were described. Investigated compounds have two values of ionisation constants except the nitro derivatives, this is the confirmation of equilibrium 3-hydroxy-1,2-benzisoxazole <==> 1,2-benzisoxazolinone-3.

Alkylation↗

The regulation of flowering time by daylength in Arabidopsis.

We are studying several Arabidopsis mutants that show altered regulation of flowering time in response to daylength. One of the mutations we are studying, constans, delays flowering under long days but has no effect under short days. Analysis of the expression of the CONSTANS gene, and modification of its expression in transgenic plants, suggests that this gene promotes flowering in response to long days and that the delay in flowering that occurs in wild-type plants under short days is at least in part due to regulation of CONSTANS gene transcription. We describe genetic approaches that we are taking to identify genes that act in the same genetic pathway as CONSTANS, and in particular the relationship between CONSTANS and two other genes that we are studying. These are LATE ELONGATED HYPOCOTYL, for which we have a dominant mutant allele that causes late flowering, and EARLY SHORT DAYS 4, whose inactivation causes early flowering. In addition to their effects on flowering time, the over-expression of CONSTANS and the inactivation of EARLY SHORT DAYS 4 cause the Arabidopsis shoot to become determinate and therefore to terminate development prematurely. This phenotype is discussed in light of other genes that have previously been shown to be required to maintain indeterminate development of the shoot.

Arabidopsis↗

Dissection of floral induction pathways using global expression analysis.

Flowering of the reference plant Arabidopsis thaliana is controlled by several signaling pathways, which converge on a small set of genes that function as pathway integrators. We have analyzed the genomic response to one type of floral inductive signal, photoperiod, to dissect the function of several genes transducing this stimulus, including CONSTANS, thought to be the major output of the photoperiod pathway. Comparing the effects of CONSTANS with those of FLOWERING LOCUS T, which integrates inputs from CONSTANS and other floral inductive pathways, we find that expression profiles of shoot apices from plants with mutations in either gene are very similar. In contrast, a mutation in LEAFY, which also acts downstream of CONSTANS, has much more limited effects. Another pathway integrator, SUPPRESSOR OF OVEREXPRESSION OF CO 1, is responsive to acute induction by photoperiod even in the presence of the floral repressor encoded by FLOWERING LOCUS C. We have discovered a large group of potential floral repressors that are down-regulated upon photoperiodic induction. These include two AP2 domain-encoding genes that can repress flowering. The two paralogous genes, SCHLAFMUTZE and SCHNARCHZAPFEN, share a signature with partial complementarity to the miR172 microRNA, whose precursor we show to be induced upon flowering. These and related findings on SPL genes suggest that microRNAs play an important role in the regulation of flowering.

Animals↗

Activation tagging of a gene for a protein with novel class of CCT-domain activates expression of a subset of sugar-inducible genes in Arabidopsis thaliana.

In the current studies, we examined sugar-inducible gene expression using the Arabidopsis thaliana line sGsL, which carries luciferase (LUC) and beta-glucuronidase (GUS) reporter genes under the control of a 210-bp promoter derived from the sweet potato sporamin gene (Spo(min)). We isolated an enhancer activation-tagged mutant of this line that showed high-level expression of LUC and GUS under non-inducing low-sugar conditions. The Activator ofSpo(min)::LUC2 (ASML2) gene located close to the enhancer encodes a protein belonging to a previously uncharacterized class of CCT (CONSTANS, CONSTANS-like, TOC1) domain proteins. Overexpression of ASML2 cDNA in the sGsL line resulted in enhanced expression of not only LUC and GUS reporters but also several endogenous sugar-inducible genes, including Atbeta-Amy, ApL3, and VSP2. Transient co-expression of 35S::ASML2 with the Spo(min)::LUC or Atbeta-Amy::LUC reporter in protoplasts resulted in an approximately 2.4 or 5.6-fold transactivation of LUC expression, respectively. Expression of ASML2 was high in reproductive organs, and expression in seedlings was slightly enhanced by sugars, but not by abscisic acid. These results suggest that ASML2 functions as a transcriptional activator and regulates the expression of at least a subset of sugar-inducible genes.

Amino Acid Sequence↗

Beyond the CO-FT regulatory module: E1 and PHYA emerge as players in photoperiodic regulation of flowering in legumes.

The legume family (Fabaceae) is the third largest in plants and includes several crop species that are able to fix nitrogen, promote soil health, and contribute to food security worldwide. Recent progress in legume genetics and genomics allowed the identification of photoperiod-dependent flowering loci, which were incorporated into specific signalling networks. Functional characterization of these regulators revealed new roles for known photoreceptors such as phytochrome A, and it also identified legume-specific B3 domain transcriptional factors (E1 and E1-like proteins). This suggests some diversification from the traditional CONSTANS-FLOWERING LOCUS T module present in other angiosperms. Although most of the findings discussed herein pertain to species from the two main legume clades, the Galegoids (e.g. alfalfa, clover, and pea) and the Phaseoloids (e.g. common bean, soybean, cowpea, and pigeon pea), research on flowering regulation in the basal Genistoid clade (e.g. lupins) will also be addressed. We propose that functional diversification of photoperiod-dependent flowering strategies in the different legume species could have contributed to their environmental adaptation and allowed their geographical expansion and success worldwide.

Photoperiod↗

A comparison between electrokinetic capillary chromatography and absorption spectroscopy for the analysis of peptide-micelle association by weak hydrophobic interactions.

Micellar electrokinetic capillary chromatography (MEKC) was compared to absorption spectroscopy to estimate equilibrium association constans (K(as)) for peptide-micelle systems involving three peptides (leucine-enkephalin, methionine-enkephalin and leucine-phenylalanine (LF)) and two surfactant micelles (sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB)). Buffer pH was chosen to minimize purely electrostatic interactions between peptides and micelles that could not be interrogated by absorption spectroscopy. Viscosity-corrected MEKC mobilities gave reasonably similar estimates of K(as) between the two methods for all three peptide-SDS micelle systems, with K(as) values ranging from 13.7 +/- 0.3 to 49 +/- 1 M(-1). For CTAB, estimates of K(as) for LF-CTAB micelle association were of the same order of magnitude as the SDS micelle by the two methods of estimation. On the other hand, enkephalin-CTAB micelle binding was about 10 times stronger (K(as) = 122 +/- 3 M(-1) to 311 +/- 9 M(-1)) than the enkephalin-SDS micelle binding. In addition, MEKC underestimated the K(as) values relative to spectroscopy by a factor of 2-3 for the enkephalin-CTAB system.

Buffers↗

Description of six new Gc variants.

Gc subtyping by isoelectric focusing and routine conventional electrophoresis demonstrated six new variants (mutants): Gc1C16, Gc1C23, Gc1C25, Gc2A12, Gc2A13, and Gc2C7. The variants were confirmed by the acknowledged Gc Reference Laboratories of Drs. J. Constans and H. Cleve. Gc1C16, Gc1C23, and Gc2A12 are tentative variants due to absence of family data to confirm their heritability.

Adult↗

Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family.

Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes, such as the establishment of floral organ identity, flowering time, and fruit development. It appears that the MADS-box gene family has undergone considerable gene duplication and divergence within various angiosperm lineages. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)/Tomato MADS-box gene 3 (TM3)-like genes are members of the MADS-box gene family and have undergone repeated duplication events. Here, we isolated and characterized the SOC1/TM3-like gene TrcMADS1 from Trillium camtschatcense (Trilliaceae) to infer the ancestral function of SOC1/TM3-like genes. The alignment of SOC1/TM3-like genes revealed the presence of a highly conserved region in the C-terminal of predicted protein sequences, designated the SOC1 motif. Phylogenetic analysis indicated that TrcMADS1 is at the basal position of the SOC1/TM3-like gene family. The TrcMADS1 mRNA was detected in both vegetative and reproductive organs by RT-PCR. Our results suggest that duplicated copies of SOC1/TM3-like gene evolved to become variously functionally specialized.

Amino Acid Sequence↗

Discovery of novel conserved peptide domains by ortholog comparison within plant multi-protein families.

Assigning individual functions to the proteins encoded by the genome of the dicotyledonous reference species Arabidopsis thaliana is one of the major challenges in current plant molecular biology. Frequently, Arabidopsis protein families are biocomputationally analyzed by multiple amino acid sequence alignments of the respective family members for detection of conserved peptide motifs that might be of functional relevance. Mere sequence alignment of paralogous sequences may obscure amino acid patches that are highly conserved amongst orthologs and thus potentially relevant for isoform-specific protein function(s). Here I exemplarily illustrate this potential pitfall by amino acid sequence alignments of the heptahelical MLO proteins using either the suite of 15 isoforms (paralogs) encoded by the Arabidopsis genome or a collection of 13 ortholog sequences derived from a set of both monocotyledonous and dicotyledonous plant species. The findings are corroborated by an analogous analysis of the distinct plant multi-protein family of CONSTANS-like transcription regulators. The data reveal that the generally higher sequence similarity of orthologs versus paralogs is not uniformly distributed among the amino acid positions of the orthologs but at least partially clustered in distinct sites/domains, suggesting conservation of isoform-specific functional modules across taxa.

Amino Acid Sequence↗

Photoperiod-regulated expression of the PpCOL1 gene encoding a homolog of CO/COL proteins in the moss Physcomitrella patens.

The CONSTANS (CO) protein is a critical regulator of the photoperiodic control of flowering in Arabidopsis thaliana and Oryza sativa. We isolated a cDNA PpCOL1 encoding a homolog of the CO/CO-LIKE (COL) family proteins from a cryptogam Physcomitrella patens. The predicted PpCOL1 protein has N-terminal zinc finger and C-terminal CCT domains, which are conserved in the angiosperm CO/COL proteins. Structurally, PpCOL1 is the most closely related to the Group Ia or Ic proteins, which include AtCO and AtCOL1/2, among diverged members of the family. A transient expression assay using GFP showed that the CCT domain of PpCOL1 contains a nuclear-localizing signal. Northern blotting analyses revealed that the PpCOL1 expression is controlled by the circadian clock, and moreover, it is photoperiodically regulated at a gametophore stage when the rate of sporophyte formation is affected by day length. These observations indicate a possible involvement of PpCOL1 as a nuclear factor in the photoperiodic regulation of reproduction of Physcomitrella.

Amino Acid Sequence↗

Photoperiodic control of flowering: not only by coincidence.

The timing of floral transition has a direct impact on reproductive success. One of the most important environmental factors that affect the transition is the change in day length (photoperiod). Classical experiments imply that plants monitor photoperiods in the leaf, and transmit that information coded within an elusive signal dubbed florigen to the apex to reprogram development. Recent advances in Arabidopsis research indicate that the core of the day-length measurement mechanism lies in the circadian regulation of CONSTANS (CO) expression and the subsequent photoperiodic induction of the expression of FLOWERING LOCUS T (FT) gene, which might encode a major component of florigen. In this review, we introduce current perspectives on how, when and where the floral signal is generated.

Flowers↗

Sequence divergence at the putative flowering time locus COL1 in Brassicaceae.

An insertion/deletion polymorphism (Ind2) in the Brassica nigra CONSTANS LIKE 1 (Bni COL1) gene was previously found to be associated with variation in flowering time. In the present study we examine the inter-specific divergence of COL1 in the family Brassicaceae. Analysis of codon substitution models did not reveal evidence of positive Darwinian selection, but comparisons of the COL1 gene in different species revealed a surprising number of indels. A total of 24 indels were found in the 650 bp of the middle variable region of the gene. This high number of indels could reflect a lack of constraint on length of this region of the protein, or the effect of positive selection. The number of indels was close to that expected in non-coding DNA, but the indels were longer in COL1 than those observed in non-coding regions. Reconstruction of indel evolution indicated that most indels resulted from deletions rather than insertions. The Ind2 indel that has shown association with flowering time in Brassica nigra exhibited a remarkable distribution in the Brassicaceae family, indicating that the polymorphism may have persisted more than ten million years. Considering presumed historic populations sizes of Brassicaceae species, such a long persistence time seems unlikely for a neutral polymorphism.

Arabidopsis Proteins↗

Antisense suppression of the Arabidopsis PIF3 gene does not affect circadian rhythms but causes early flowering and increases FT expression.

Photoperiodic control of flowering is regulated by light and a circadian clock. Feedback regulation of the transcription of clock components is one of the most common and important mechanisms that control clock functions in animals, fungi, and plants. The Arabidopsis circadian clock is believed to involve two myb-related proteins, LHY (late elongated hypocotyl) and CCA1 (circadian clock associated 1), which negatively regulate TOC1 (timing of cab expression 1) gene expression through direct binding to the TOC1 promoter. PIF3 (phytochrome-interacting factor 3), a bHLH transcription factor binds promoter regions of the LHY and CCA1 genes, affecting the light induction of these genes, and interacts with TOC1 protein. Although the positive feedback regulation of clock components in plants has been predicted, and PIF3 has been assumed to be involved, the molecular nature of this process has not been elucidated. Here we demonstrate that the antisense suppression of the PIF3 gene causes higher levels of mRNA of floral activator genes CO (constans) and FT (flowering locus T) and results in early flowering under long days (LD). Neither the circadian rhythms of the clock-controlled genes (CCGs) under constant conditions nor the diurnal rhythms of the CCGs under LD conditions are affected by the reduction in PIF3 gene expression. These results suggest that PIF3 may play an important role in the control of flowering through clock-independent regulation of CO and FT gene expression in Arabidopsis.

Arabidopsis↗

Photoperiodism: the coincidental perception of the season.

Many plants use the seasonal change in daylength as a signal for flowering. Daylength sensing in Arabidopsis has now been shown to occur by an external coincidence mechanism, which operates by the circadian and light regulation of CONSTANS.

Arabidopsis↗