Ilha Lee

Analysis of Transcription Factor HY5 Genomic Binding Sites Revealed Its Hierarchical Role in Light Regulation of Development

The transcription factor LONG HYPOCOTYL5 (HY5) acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here, we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using a high-density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis thaliana genome, we mapped genome-wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed >3000 chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light-responsive genes and transcription factor genes. Our data thus support a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis.

A genetic framework for floral patterning

The initial steps of flower development involve two classes of consecutively acting regulatory genes. Meristem-identity genes, which act early to control the initiation of flowers, are expressed throughout the incipient floral primordium. Homeotic genes, which act later to specify the identity of individual floral organs, are expressed in distinct domains within the flower. The link between the two classes of genes has remained unknown so far. Here we show that the meristem-identity gene LEAFY has a role in controlling homeotic genes that is separable from its role in specifying floral fate. On the basis of our observation that LEAFY activates different homeotic genes through distinct mechanisms, we propose a genetic framework for the control of floral patterning.

Isolation of LUMINIDEPENDENS: A Gene Involved in the Control of Flowering Time in Arabidopsis

Plants have evolved the ability to regulate flowering in response to environmental signals such as temperature and photoperiod. The physiology and genetics of floral induction have been studied extensively, but the molecular mechanisms that underlie this process are poorly understood. To study this process, we isolated a gene, LUMINIDEPENDENS (LD), that is involved in the timing of flowering in Arabidopsis. Mutations in this gene render Arabidopsis late flowering and appear to affect light perception. The late-flowering phenotype of the ld mutation was partially suppressed by vernalization. Genomic and cDNA clones of the LD gene were characterized. The predicted amino acid sequence of the LD protein contains 953 residues and includes two putative bipartite nuclear localization signals and a glutamine-rich region.

Regulation and function of SOC1, a flowering pathway integrator

SOC1, encoding a MADS box transcription factor, integrates multiple flowering signals derived from photoperiod, temperature, hormone, and age-related signals. SOC1 is regulated by two antagonistic flowering regulators, CONSTANS (CO) and FLOWERING LOCUS C (FLC), which act as floral activator and repressor, respectively. CO activates SOC1 mainly through FT but FLC represses SOC1 by direct binding to the promoter. SOC1 is also activated by an age-dependent mechanism in which SPL9 and microRNA156 are involved. When SOC1 is induced at the shoot apex, SOC1 together with AGL24 directly activates LEAFY (LFY), a floral meristem identity gene. APETALA1 (AP1), activated mainly by FT, is also necessary to establish and maintain flower meristem identity. When LFY and AP1 are established, flower development occurs at the anlagen of shoot apical meristem according to the ABC model. During early flower development, AP1 activates the A function and represses three redundantly functioning flowering time genes, SOC1, AGL24, and SVP to prevent floral reversion. During late flower development, such repression is also necessary to activate SEPALATA3 (SEP3) which is a coactivator of B and C function genes with LFY, otherwise SEP3 is suppressed by SOC1, AGL24, and SVP. Therefore, SOC1 is necessary to prevent premature differentiation of the floral meristem.

A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana

Cold induces expression of a number of genes that encode proteins that enhance tolerance to freezing temperatures in plants. A cis-acting element responsive to cold and drought, the C-repeat/dehydration-responsive element (C/DRE), was identified in the Arabidopsis thaliana stress-inducible genes RD29A and COR15a and found in other cold-inducible genes in various plants. C/DRE-binding factor/DRE-binding protein (CBF/DREB) is an essential component of the cold-acclimation response, but the signaling pathways and networks are mostly unknown. Here we used targeted genetic approach to isolate A. thaliana mutants with altered cold-responsive gene expression (acg) and identify ACG1 as a negative regulator of the CBF/DREB pathway. acg1 flowered late and had elevated expression of FLOWERING LOCUS C (FLC), a repressor of flowering encoding a MADS-box protein. We showed that acg1 is a null allele of the autonomous pathway gene FVE. FVE encodes a homolog of the mammalian retinoblastoma-associated protein, a component of a histone deacetylase (HDAC) complex involved in transcriptional repression. We also showed that plants sense intermittent cold stress through FVE and delay flowering with increasing expression of FLC. Dual roles of FVE in regulating the flowering time and the cold response may have an evolutionary advantage for plants by increasing their survival rates.

A LEAFY co-regulator encoded by UNUSUAL FLORAL ORGANS

BACKGROUND . Development of petals and stamens in Arabidopsis flowers requires the function of the organ-identity gene APETALA3 (AP3), whose RNA is expressed specifically in petal and stamen primordia. AP3 expression is positively regulated by the meristem-identity gene LEAFY (LFY), which is expressed ubiquitously in young flowers. It is unknown how the transition from ubiquitous expression of LFY to region-specific expression of AP3 is made. It has previously been proposed for Antirrhinum that another gene, FIMBRIATA (FIM), mediates between the LFY and AP3 orthologs, with the three genes acting in a simple regulatory hierarchy. FIM is activated later than the LFY ortholog, and its expression is more restricted than that of the LFY ortholog. RESULTS . We have tested whether the model proposed for Antirrhinum applies to Arabidopsis, by creating transgenic plants in which the FIM ortholog UNUSUAL FLORAL ORGANS (UFO) was expressed constitutively from the promoter of the cauliflower mosaic virus 35S gene. In 35S::UFO flowers, AP3 was expressed precociously and ectopically, confirming that UFO is an upstream regulator of AP3. However, 35S::UFO could not restore petal and stamen development in lfy mutants, indicating that UFO can only function in the presence of LFY activity. The failure of 35S::UFO to rescue lfy mutants is consistent with our observation that UFO expression levels are not markedly changed in lfy mutants. CONCLUSIONS . We conclude that UFO is not a simple mediator between meristem- and organ-identity genes, but is likely to be a partially dispensable co-regulator that acts together with LFY. The interplay between LFY and UFO provides a paradigm for how a global regulator such as LFY activates selected target genes only in restricted regions within its expression domain.

COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability

Seasonal changes in day length are perceived by plant photoreceptors and transmitted to the circadian clock to modulate developmental responses such as flowering time. Blue-light-sensing cryptochromes, the E3 ubiquitin-ligase COP1, and clock-associated proteins ELF3 and GI regulate this process, although the regulatory link between them is unclear. Here we present data showing that COP1 acts with ELF3 to mediate day length signaling from CRY2 to GI within the photoperiod flowering pathway. We found that COP1 and ELF3 interact in vivo and show that ELF3 allows COP1 to interact with GI in vivo, leading to GI degradation in planta. Accordingly, mutation of COP1 or ELF3 disturbs the pattern of GI cyclic accumulation. We propose a model in which ELF3 acts as a substrate adaptor, enabling COP1 to modulate light input signal to the circadian clock through targeted destabilization of GI.

SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy.

SUMMARY Suppressor of overexpression of constans1 (SOC1) is one of the flowering pathway integrators and regulates the expression of LEAFY (LFY), which links floral induction and floral development. However, the mechanism by which SOC1, a MADS box protein, regulates LFY has proved elusive. Here, we show that SOC1 directly binds to the distal and proximal region of the LFY promoter where critical cis-elements are located. Intragenic suppressor mutant analysis shows that a missense mutation in the MADS box of SOC1 causes loss of binding to the LFY promoter as well as suppression of the flowering promotion function. The full-length SOC1 protein locates in the cytoplasm if expressed alone in protoplast transient expression assay, but relocates to the nucleus if expressed with AGAMOUS-LIKE 24 (AGL24), another flowering pathway integrator and a MADS box protein. The domain analysis shows that co-localization of SOC1 and AGL24 is mediated by the MADS box and the intervening region of SOC1. Finally, we show that LFY is expressed only in those tissues where SOC1 and AGL24 expressions overlap. Thus, we propose that heterodimerization of SOC1 and AGL24 is a key mechanism in activating LFY expression.

A new arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.

The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure

Loss-of-function siz1 mutations caused early flowering under short days. siz1 plants have elevated salicylic acid (SA) levels, which are restored to wild-type levels by expressing nahG, bacterial salicylate hydroxylase. The early flowering of siz1 was suppressed by expressing nahG, indicating that SIZ1 represses the transition to flowering mainly through suppressing SA-dependent floral promotion signaling under short days. Previous results have shown that exogenous SA treatment does not suppress late flowering of autonomous pathway mutants. However, the siz1 mutation accelerated flowering time of an autonomous pathway mutant, luminidependens, by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor. This result suggests that SIZ1 promotes FLC expression, possibly through an SA-independent pathway. Evidence indicates that SIZ1 is required for the full activation of FLC expression in the late-flowering FRIGIDA background. Interestingly, increased FLC expression and late flowering of an autonomous pathway mutant, flowering locus d (fld), was not suppressed by siz1, suggesting that SIZ1 promotes FLC expression by repressing FLD. Consistent with this, SIZ1 facilitates sumoylation of FLD that can be suppressed by mutations in three predicted sumoylation motifs in FLD (i.e. FLDK3R). Furthermore, expression of FLDK3R in fld protoplasts strongly reduced FLC transcription compared with expression of FLD, and this affect was linked to reduced acetylation of histone 4 in FLC chromatin. Taken together, the results suggest that SIZ1 is a floral repressor that not only represses the SA-dependent pathway, but also promotes FLC expression by repressing FLD activity through sumoylation, which is required for full FLC expression in a FRIGIDA background.