Antagonistic roles of SEPALLATA3, FT and FLC genes as targets of the polycomb group gene CURLY LEAF

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering.     

Interaction of Polycomb-group proteins controlling flowering in Arabidopsis

In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.     

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering

Flowering in Arabidopsis thaliana is promoted by long-day (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 ( elf 3) mutant of Arabidopsis , which is insensitive to photoperiod with regard to floral initiation has been characterized. elf 3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf 3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf 3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY 4. However, when elf 3 mutants were made deficient for functional phytochrome by the introduction of hy 2 mutant alleles, the elf 3 hy 2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf 3 single mutants. In addition, the inflorescences of one allelic combination of elf 3 hy 2 double mutants form a terminal flower similar to the structure produced by tfl 1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.     

Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components

Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

Reprogramming of H3K27me3 Is Critical for Acquisition of Pluripotency from Cultured Arabidopsis Tissues

In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn) and embryonic flower2 (emf2) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP-chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> gene that is involved in the control of cell differentiation in leaves

During leaf development, the formation of dorsal-ventral and proximal-distal axes is central to leaf morphogenesis. To investigate the genetic basis of dorsoventrality and proximodistality in the leaf, we screened for mutants of Arabidopsis thaliana (L.) Heynh. with defects in leaf morphogenesis. We describe here the phenotypic analysis of three mutant alleles that we have isolated. These mutants show varying degrees of abnormality including dwarfism, broad leaf lamina, and aberrant floral organs and fruits. Genetic analysis revealed that these mutations are alleles of the previously isolated mutant asymmetric leaves1 ( as1). In addition to the leaf phenotypes described previously, these alleles display other phenotypes that were not observed. These include: (i) some rosette leaves with petiole growth underneath the leaf lamina; (ii) leaf vein branching in the petiole; and (iii) a leaf lamina with an epidermis similar to that on the petiole. The mutant phenotypes suggest that the ASYMMETRIC LEAVES1 ( AS1) gene is involved in the control of cell differentiation in leaves. As the first step in determining a molecular function for AS1, we have identified the AS1 gene using map-based cloning. The AS1 gene encodes a MYB-domain protein that is homologous to the Antirrhinum PHANTASTICA ( PHAN) and maize ROUGH SHEATH2 ( RS2) genes. AS1 is expressed nearly ubiquitously, consistent with the pleiotropic mutant phenotypes. High levels of AS1 expression were found in tissues with highly proliferative cells, which further suggests a role in cell division and early cell differentiation.     

Monogenic Recessive Mutations Causing Both Late Floral Initiation and Excess Starch Accumulation in Arabidopsis

A recessive Arabidopsis mutation, carbohydrate accumulation mutant1 (cam1), which maps to position 22.8 on chromosome 3, was identified by screening leaves of ethyl methanesulfonate-mutagenized M2 plants stained with iodine for altered starch content. Increased starch content in leaves of the cam1 mutant was observed at the onset of flowering. This mutant also had a delayed floral initiation phenotype with more rosette leaves than the parental line. In addition, activities of several enzymes associated with starch metabolism were altered in the cam1 mutant. The late-flowering mutant gigantea (gi) also manifested an elevated starch level in leaves. However, not all late-flowering mutants had increased leaf starch content. Double mutants cam1 adg1 (for ADP-glucose pyrophosphorylase), cam1 pgm (for phosphoglucomutase), and gi pgm had no observable starch in leaves but showed the late-flowering phenotype, demonstrating that the elevated starch content is not the cause of late floral initiation. The pleiotropic effects of cam1 and gi suggest that they may play regulatory roles in starch metabolism and floral initiation. These data suggest that starch accumulation and floral initiation may share a common regulatory pathway.