Search

Allelic variation in FLOWERING LOCUS C (FLC), a central repressor of flowering, contributes to natural differences in flowering behavior among Arabidopsis accessions. The weak nature of the FLC allele in the Ler accession is due to low levels of FLC RNA resulting, through an unknown mechanism, from a transposable element inserted in an intron of FLC. Here we show that the transposable element renders FLC-Ler subject to repressive chromatin modifications mediated by short interfering RNAs generated from homologous transposable elements in the genome. Our studies have general implications for the role of transposable elements in eukaryotic gene expression and evolution.

The winter-annual habit (which typically involves a requirement for exposure to the cold of winter to flower in the spring) in Arabidopsis thaliana is mainly due to the repression of flowering by relatively high levels of FLC expression. Exposure to prolonged cold attenuates FLC expression through a process known as vernalization and thus permits flowering to occur in the spring. Here we show that the elevated FLC expression characteristic of nonvernalized winter annuals requires two genes, EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING 8 (ELF8), that are homologs of components of the PAF1 complex of Saccharomyces cerevisiae. Furthermore, ELF7 and ELF8 are also required for the expression of other genes in the FLC clade of flowering repressors such as MAF2 and FLM. FLC, FLM, and MAF2 are involved in multiple flowering pathways that account for the broad effects of elf7 and elf8 mutations on flowering behavior. ELF7 and ELF8 are required for the enhancement of histone 3 trimethylation at Lys 4 in FLC chromatin. This modification of FLC chromatin appears to be required to elevate FLC expression to levels that can delay flowering in plants that have not been vernalized. A model of the role of ELF7, ELF8, and other previously described genes in the modification of the chromatin of flowering repressors is presented.

Recessive mutations that suppress the late-flowering phenotype conferred by FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) and which also result in serrated leaf morphology were identified in T-DNA and fast-neutron mutant populations. Molecular analysis showed that the mutations are caused by lesions in the gene encoding the large subunit of the nuclear mRNA cap-binding protein, ABH1 (ABA hypersensitive1). The suppression of late flowering is caused by the inability of FRI to increase FLC mRNA levels in the abh1 mutant background. The serrated leaf morphology of abh1 is similar to the serrate (se) mutant and, like abh1, se is also a suppressor of FRI-mediated late flowering although it is a weaker suppressor than abh1. Unlike se, in abh1 the rate of leaf production and the number of juvenile leaves are not altered. The abh1 lesion affects several developmental processes, perhaps because the processing of certain mRNAs in these pathways is more sensitive to loss of cap-binding activity than the majority of cellular mRNAs.

A small organizing center, the quiescent center (QC), maintains stem cells in the Arabidopsis root and defines the stem cell niche. The phytohormone auxin influences the position of this niche by an unknown mechanism. Here, we identify the PLETHORA1 (PLT1) and PLT2 genes encoding AP2 class putative transcription factors, which are essential for QC specification and stem cell activity. The PLT genes are transcribed in response to auxin accumulation and are dependent on auxin response transcription factors. Distal PLT transcript accumulation creates an overlap with the radial expression domains of SHORT-ROOT and SCARECROW, providing positional information for the stem cell niche. Furthermore, the PLT genes are activated in the basal embryo region that gives rise to hypocotyl, root, and root stem cells and, when ectopically expressed, transform apical regions to these identities. Thus, the PLT genes are key effectors for establishment of the stem cell niche during embryonic pattern formation.

Flowering in Arabidopsis thaliana is controlled by multiple pathways, including the photoperiod pathway and the FLOWERING LOCUS C (FLC)-dependent pathway. Here, we report that a pair of related jumonji-class transcription factors, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play divergent roles in the regulation of Arabidopsis flowering. ELF6 acts as a repressor in the photoperiod pathway, whereas REF6, which has the highest similarity to ELF6, is an FLC repressor. Ectopic expression studies and expression pattern analyses show that ELF6 and REF6 have different cellular roles and are also regulated differentially despite their sequence similarities. Repression of FLC expression by REF6 accompanies histone modifications in FLC chromatin, indicating that the transcriptional regulatory activity of this class of proteins includes chromatin remodeling. This report demonstrates the in vivo functions of this class of proteins in higher eukaryotes.

EARLY FLOWERING 5 (ELF5) is a single-copy gene involved in flowering time regulation in Arabidopsis. ELF5 encodes a nuclear-targeted protein that is related to the human nuclear protein containing a WW domain (Npw)38-binding protein (NpwBP). Lesions in ELF5 cause early flowering in both long days and short days. elf5 mutations partially suppress the late flowering of both autonomous-pathway mutants and FRIGIDA (FRI)-containing lines by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor upon which many of the flowering pathways converge. elf5 mutations also partially suppress photoperiod-pathway mutants, and this, along with the ability of elf5 mutations to cause early flowering in short days, indicates that ELF5 also affects flowering independently of FLC.

In temperate climates, the prolonged cold temperature of winter serves as a seasonal landmark for winter-annual and biennial plants. In these plants, flowering is blocked before winter. In Arabidopsis thaliana, natural variation in the FRIGIDA (FRI) gene is a major determinate of the rapid-cycling vs. winter-annual flowering habits. In winter-annual accessions of Arabidopsis, FRI activity blocks flowering through the up-regulation of the floral inhibitor FLOWERING LOCUS C (FLC). Most rapid-flowering accessions, in contrast, contain null alleles of FRI. By performing a mutant screen in a winter-annual strain, we have identified a locus, FRIGIDA LIKE 1 (FRL1), that is specifically required for the up-regulation of FLC by FRI. Cloning of FRL1 revealed a gene with a predicted protein sequence that is 23% identical to FRI. Despite sequence similarity, FRI and FRL1 do not have redundant functions. FRI and FRL1 belong to a seven-member gene family in Arabidopsis, and FRI, FRL1, and at least one additional family member, FRIGIDA LIKE 2 (FRL2), are in a clade of this family that is required for the winter-annual habit in Arabidopsis.

One of the remarkable aspects of the promotion of flowering by vernalization is that plants have evolved the ability to measure a complete winter season of cold and to 'remember' this prior cold exposure in the spring. Recent work in Arabidopsis demonstrates the molecular basis of this memory of winter: vernalization causes changes in the chromatin structure of a flowering repressor gene, FLOWERING LOCUS C (FLC), that switch this gene into a repressed state that is mitotically stable. A key component of the vernalization pathway, VERNALIZATION INSENSITIVE3 (VIN3), which is a PHD-domain-containing protein, is induced only after a prolonged period of cold. VIN3 is involved in initiating the modification of FLC chromatin structure. The stable silencing of FLC also requires the DNA-binding protein VERNALIZATION1 (VRN1) and the polycomb-group protein VRN2.

In biennials and winter annuals, flowering is typically blocked in the first growing season. Exposure to the prolonged cold of winter, through a process called vernalization, is required to alleviate this block and permit flowering in the second growing season. In winter-annual types of Arabidopsis thaliana, a flowering repressor, FLOWERING LOCUS C (FLC), is expressed at levels that inhibit flowering in the first growing season. Vernalization promotes flowering by causing a repression of FLC that is mitotically stable after return to warm growing conditions. Here we identify a gene with a function in the measurement of the duration of cold exposure and in the establishment of the vernalized state. We show that this silencing involves changes in the modification of histones in FLC chromatin.

The Arabidopsis autonomous floral-promotion pathway promotes flowering independently of the photoperiod and vernalization pathways by repressing FLOWERING LOCUS C (FLC), a MADS-box transcription factor that blocks the transition from vegetative to reproductive development. Here, we report that FLOWERING LOCUS D (FLD), one of six genes in the autonomous pathway, encodes a plant homolog of a protein found in histone deacetylase complexes in mammals. Lesions in FLD result in hyperacetylation of histones in FLC chromatin, up-regulation of FLC expression, and extremely delayed flowering. Thus, the autonomous pathway regulates flowering in part by histone deacetylation. However, not all autonomous-pathway mutants exhibit FLC hyperacetylation, indicating that multiple means exist by which this pathway represses FLC expression.

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

Flowering time control in plants involves integration of multiple signals. One of the signalling pathways in Arabidopsis involves a negative autoregulatory loop, in which the FCA protein together with FY promotes the choice of an alternative polyadenylation site within the FCA pre-mRNA to produce a transcript that does not encode a functional protein.

Plant species have evolved a wide variety of flowering habits, each adapted to maximize reproductive success in their local environment. Even within a species, accessions from different environments can exhibit markedly different flowering behavior. In Arabidopsis, some accessions are rapid-cycling summer annuals, whereas others accessions are late flowering and vernalization responsive and thus behave as winter annuals. Two genes, FLOWERING LOCUS C (FLC) and FRIGIDA (FRI), interact synergistically to confer the winter-annual habit. Previous work has shown that many summer-annual accessions contain null mutations in the FRI gene; thus it appears that these summer-annual accessions have arisen from winter-annual ancestors by losing FRI function. In this work we demonstrate that naturally occurring allelic variation in FLC has provided another route to the evolution of summer-annual flowering behavior in Arabidopsis. We have identified two summer-annual accessions, Da (1)-12 and Shakhdara, that contain functional alleles of FRI, but are early flowering because of weak alleles of FLC. We have also determined that the weak allele of FLC found in Landsberg erecta is naturally occurring. Unlike accessions that have arisen because of loss-of-function mutations in FRI, the FLC alleles from Da (1)-12, Shakhdara, and Landsberg erecta are not nulls; however, they exhibit lower steady-state mRNA levels than strong alleles of FLC. Sequence analysis indicates that these weak alleles of FLC have arisen independently at least twice during the course of evolution.

Proper control of the floral transition is critical for reproductive success in flowering plants. In Arabidopsis, FLOWERING LOCUS C (FLC) is a floral repressor upon which multiple floral regulatory pathways converge. Mutations in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) suppress the FLC-mediated delay of flowering as a result of the presence of FRIGIDA or of mutations in autonomous pathway genes. PIE1 is required for high levels of FLC expression in the shoot apex, but it is not required for FLC expression in roots. PIE1 is similar to ATP-dependent, chromatin-remodeling proteins of the ISWI and SWI2/SNF2 family. The role of PIE1 as an activator of FLC is consistent with the general role of ISWI and SWI2/SNF2 family genes as activators of gene expression. The pie1 mutation also causes early flowering in noninductive photoperiods independently of FLC; thus, PIE1 appears to be involved in multiple flowering pathways. PIE1 also plays a role in petal development, as revealed by the suppression of petal defects of the curly leaf mutant by the pie1 mutation.

FLOWERING LOCUS M (FLM) is a MADS-domain gene that acts as an inhibitor of flowering in Arabidopsis. Here we describe the genetic interaction of FLM with genes in the photoperiod and autonomous flowering pathways. Although the sequence of FLM is most similar to that of FLC, FLM and FLC interact with different flowering pathways. It has been previously shown that flc lesions suppress the late-flowering phenotype of FRI-containing lines and autonomous-pathway mutants. However, flm lesions suppress the late-flowering phenotype of photoperiod-pathway mutants but not that of FRI-containing lines or autonomous-pathway mutants. Another MADS-domain flowering repressor with a mutant phenotype similar to FLM is SVP. The late-flowering phenotype of FLM over-expression is suppressed by the svp mutation, and an svp flm double mutant behaves like the single mutants. Thus FLM and SVP are in the same flowering pathway which interacts with the photoperiod pathway.

MADS-domain-containing transcription factors comprise a large family of regulators that have diverse roles in plant development, including the regulation of flowering time. AGAMOUS-LIKE 20/SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFUL act to promote flowering, whereas FLOWERING LOCUS C (FLC), FLOWERING LOCUS M/MADS AFFECTING FLOWERING1, and SHORT VEGETATIVE PHASE are inhibitors of flowering. Here we report that AGAMOUS-LIKE 24 (AGL24) also plays a role in the regulation of flowering time. agl24 mutants are late flowering and overexpression of AGL24 causes early flowering in wild-type and late-flowering-mutant backgrounds. The effect of AGL24 overexpression is most pronounced in autonomous-pathway-mutant and FRIGIDA-containing backgrounds. The behavior of AGL24 is most similar to that of SOC1. Like SOC1, AGL24 mRNA levels are upregulated by vernalization. Unlike SOC1, however, AGL24 mRNA levels are not affected by FLC, and therefore AGL24 may represent an FLC-independent target of the vernalization pathway. There is also evidence for cross-talk between AGL24 and SOC1. When overexpressed, SOC1 and AGL24 are able to upregulate each other's expression. Thus, AGL24 represents another component in a network of MADS-domain-containing transcription factors that regulate flowering time.

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.

Functional genetic redundancy is widespread in plants and could have an important impact on phenotypic diversity if the multiple gene copies act in an additive or dosage-dependent manner. We have cloned four Brassica rapa homologs (BrFLC) of the MADS-box flowering-time regulator FLC, located at the top of chromosome 5 of Arabidopsis thaliana. Relative rate tests revealed no evidence for differential rates of evolution and the ratios of nonsynonymous-to-synonymous substitutions suggest BrFLC loci are not under strong purifying selection. BrFLC1, BrFLC2, and BrFLC3 map to genomic regions that are collinear with the top of At5, consistent with a polyploid origin. BrFLC5 maps near a junction of two collinear regions to Arabidopsis, one of which includes an FLC-like gene (AGL31). However, all BrFLC sequences are more closely related to FLC than to AGL31. BrFLC1, BrFLC2, and BrFLC5 cosegregate with flowering-time loci evaluated in populations derived by backcrossing late-flowering alleles from a biennial parent into an annual parent. Two loci segregating in a single backcross population affected flowering in a completely additive manner. Thus, replicated BrFLC genes appear to have a similar function and interact in an additive manner to modulate flowering time.

Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.