CURLY LEAF Research Articles

The CHD3 Chromatin Remodeler PICKLE and Polycomb Group Proteins Antagonistically Regulate Meristem Activity in the Arabidopsis Root

The chromatin modifying Polycomb group (PcG) and trithorax group (trxG) proteins are central regulators of cell identity that maintain a tightly controlled balance between cell proliferation and cell differentiation. The opposing activities of PcG and trxG proteins ensure the correct expression of specific transcriptional programs at defined developmental stages. Here, we report that the chromatin remodeling factor PICKLE (PKL) and the PcG protein CURLY LEAF (CLF) antagonistically determine root meristem activity. Whereas loss of PKL function caused a decrease in meristematic activity, loss of CLF function increased meristematic activity. Alterations of meristematic activity in pkl and clf mutants were not connected with changes in auxin concentration but correlated with decreased or increased expression of root stem cell and meristem marker genes, respectively. Root stem cell and meristem marker genes are modified by the PcG-mediated trimethylation of histone H3 on lysine 27 (H3K27me3). Decreased expression levels of root stem cell and meristem marker genes in pkl correlated with increased levels of H3K27me3, indicating that root meristem activity is largely controlled by the antagonistic activity of PcG proteins and PKL.

The CURLY LEAF Interacting Protein BLISTER Controls Expression of Polycomb-Group Target Genes and Cellular Differentiation ofArabidopsis thaliana

Polycomb-group (Pc-G) proteins are important regulators of many developmental processes in plants and animals and repress gene expression by imparting histone H3 lysine 27 trimethylation (H3K27me3). Here, we present the identification of the novel, plant-specific Arabidopsis thaliana protein BLISTER (BLI), which interacts with the Pc-G histone methyltransferase CURLY LEAF (CLF). We map the interaction of BLI with CLF to a predicted coiled-coil domain in BLI that shares similarity with STRUCTURAL MAINTENANCE OF CHROMOSOMES proteins. BLI colocalizes with CLF in the nucleus, shows an overlapping expression pattern with CLF throughout plant development that is strongest in dividing cells, and represses a subset of Pc-G target genes. Loss of BLI results in a pleiotropic developmental mutant phenotype, indicating that BLI prevents premature differentiation. Furthermore, bli mutants exhibit severe epidermal defects, including loss of cell adhesion, outgrowth of cells, and increased cotyledon cell size. As these phenotypes have not been observed in Pc-G mutants, we propose that BLI has functions related to Pc-G proteins but can also act independently in Arabidopsis development.

Dosage-Sensitive Function of RETINOBLASTOMA RELATED and Convergent Epigenetic Control Are Required during the Arabidopsis Life Cycle

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.

The SAND domain protein ULTRAPETALA1 acts as a trithorax group factor to regulate cell fate in plants

During development, trithorax group (trxG) chromatin remodeling complexes counteract repression by Polycomb group (PcG) complexes to sustain active expression of key regulatory genes. Although PcG complexes are well characterized in plants, little is known about trxG activities. Here we demonstrate that the Arabidopsis SAND (Sp100, AIRE-1, NucP41/75, DEAF-1) domain protein ULTRAPETALA1 (ULT1) functions as a trxG factor that counteracts the PcG-repressive activity of CURLY LEAF. In floral stem cells, ULT1 protein associates directly with the master homeotic locus AGAMOUS, inducing its expression by regulating its histone methylation status. Our analysis introduces a novel mechanism that mediates epigenetic switches controlling post-embryonic stem cell fates in plants.

A Single Amino Acid Change in the Enhancer of Zeste Ortholog CURLY LEAF Results in Vernalization-Independent, Rapid Flowering in Arabidopsis

Many strains of Arabidopsis (Arabidopsis thaliana) require exposure to prolonged cold for rapid flowering, a process known as vernalization. Vernalization in Arabidopsis results in the suppression of FLOWERING LOCUS C (FLC), a repressor of flowering. In a screen for mutants that no longer require vernalization for rapid flowering, we identified a dominant allele of the Enhancer of Zeste E(z) ortholog CURLY LEAF (CLF), clf-59. CLF is a Polycomb Group gene, and the clf-59 mutant protein contains a proline-to-serine transition in a cysteine-rich region that precedes the SET domain. Mutant plants are early flowering and have reduced FLC expression, but, unlike clf loss-of-function mutants, clf-59 mutants do not display additional pleiotropic phenotypes. clf-59 mutants have elevated levels of trimethylation on lysine 27 of histone H3 (H3K27me3) at FLC. Thus, clf-59 appears to be a gain-of-function allele, and this allele represses FLC without some of the components required for vernalization-mediated repression. In the course of this work, we also identified a marked difference in H3K27me3 levels at FLC between plants that contain and those that lack the FRIGIDA (FRI) gene. Furthermore, FRI appears to affect CLF occupancy at FLC; thus, our work provides insight into the molecular role that FRI plays in delaying the onset of flowering.

CHD3 Proteins and Polycomb Group Proteins Antagonistically Determine Cell Identity in Arabidopsis

Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG) and trithorax group (trxG) proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA–binding domain CHD3 proteins PICKLE (PKL) and PICKLE RELATED2 (PKR2) have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3). Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote cell differentiation by suppressing embryonic development.

Development of a hierarchical Bayesian model to estimate the growth parameters of Listeria monocytogenes in minimally processed fresh leafy salads

The optimal growth rate µ opt of Listeria monocytogenes in minimally processed (MP) fresh leafy salads was estimated with a hierarchical Bayesian model at (mean ± standard deviation) 0.33 ± 0.16 h − 1 . This µ opt value was much lower on average than that in nutrient broth, liquid dairy, meat and seafood products (0.7–1.3 h − 1 ), and of the same order of magnitude as in cheese. Cardinal temperatures T min, T opt and T max were determined at − 4.5 ± 1.3 °C, 37.1 ± 1.3 °C and 45.4 ± 1.2 °C respectively. These parameters were determined from 206 growth curves of L. monocytogenes in MP fresh leafy salads (lettuce including iceberg lettuce, broad leaf endive, curly leaf endive, lamb's lettuce, and mixtures of them) selected in the scientific literature and in technical reports. The adequacy of the model was evaluated by comparing observed data (bacterial concentrations at each experimental time for the completion of the 206 growth curves, mean log 10 increase at selected times and temperatures, L. monocytogenes concentrations in naturally contaminated MP iceberg lettuce) with the distribution of the predicted data generated by the model. The sensitivity of the model to assumptions about the prior values also was tested. The observed values mostly fell into the 95% credible interval of the distribution of predicted values. The µ opt and its uncertainty determined in this work could be used in quantitative microbial risk assessment for L. monocytogenes in minimally processed fresh leafy salads.

Polycomb Silencing of KNOX Genes Confines Shoot Stem Cell Niches in Arabidopsis

Most plant organs develop postembryonically from stem cells in the shoot and root meristems. In Arabidopsis, Class I KNOTTED-like homeobox (KNOX) transcription factors are specifically expressed in shoot meristems and play a primary role in the maintenance of meristem function. Although suppression of KNOX was shown to associate with histone H3K27-methylation, the molecular mechanism underlying this suppression is not well understood. Here, we provide genetic, molecular, and functional evidence that an Arabidopsis POLYCOMB REPRESSIVE COMPLEX1 (PRC1)-like complex acts in conjunction with PRC2 in KNOX suppression. We identified AtRING1a and AtRING1b as homologs of the animal PRC1 core component RING1. Loss-of-function mutant Atring1a(-/-)Atring1b(-/-) shows release of KNOX suppression and ectopic-meristem formation. AtRING1a and AtRING1b proteins are localized in the nucleus. AtRING1a binds to itself and to AtRING1b, to CURLY LEAF (CLF), a PRC2 core component catalyzing H3K27-methylation, and to LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a chromodomain protein binding trimethyl-H3K27. We further show that clf(-/-) and lhp1(-/-) enhance Atring1a(-/-)Atring1b(-/-) in release of KNOX suppression and mutant phenotypes. We propose a model in which AtRING1a, AtRING1b, and LHP1 form a PRC1-like complex, which binds trimethyl-H3K27 marked by the CLF-containing PRC2, resulting in transcriptional suppression of KNOX.

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.

Sexual and Apomictic Seed Formation in Hieracium Requires the Plant Polycomb-Group Gene FERTILIZATION INDEPENDENT ENDOSPERM

A Polycomb-Group (PcG) complex, FERTILIZATION INDEPENDENT SEED (FIS), represses endosperm development in Arabidopsis thaliana until fertilization occurs. The Hieracium genus contains apomictic species that form viable seeds asexually. To investigate FIS function during apomictic seed formation, FERTILIZATION INDEPENDENT ENDOSPERM (FIE), encoding a WD-repeat member of the FIS complex, was isolated and downregulated in sexual and apomictic Hieracium species. General downregulation led to defects in leaf and seed development, consistent with a role in developmental transitions and cell fate. PcG-like activity of Hieracium FIE was also supported by its interaction in vitro with the Arabidopsis CURLY LEAF PcG protein. By contrast, specific downregulation of FIE in developing seeds of sexual Hieracium did not result in autonomous endosperm proliferation but led to seed abortion after cross-pollination. Furthermore, in apomictic Hieracium, specific FIE downregulation inhibited autonomous embryo and endosperm initiation, and most autonomous seeds displayed defective embryo and endosperm growth. Therefore, FIE is required for both apomictic and fertilization-induced seed initiation in Hieracium. Since Hieracium FIE failed to interact with FIS class proteins in vitro, its partner proteins might differ from those in the FIS complex of Arabidopsis. These differences in protein interaction were attributed to structural modifications predicted from comparisons of Arabidopsis and Hieracium FIE molecular models.

A gain-of-function mutation of transcriptional factor PTL results in curly leaves, dwarfism and male sterility by affecting auxin homeostasis

GT factors are plant-specific trihelix DNA-binding transcription factors, which are involved in light responses and other developmental processes in plant. We identified a gain-of-function mutant of a GT-2 factor gene, PETAL LOSS (PTL), which displayed pleiotropic phenotypes including dwarfism, curly leaves, retarded growth and male sterility. We found that constitutive and ectopic over-expression of PTL driven by the CaMV 35S promoter could not recapitulate the phenotypes of the 35S enhancer-driven mutant ptl-D, and was lethal in some of the transgenic plants at the cotyledon developmental stage, suggesting that accurate temporal and spatial expression of PTL is essential for its proper functional implementation during plant development. Further analysis showed that ptl-D was defective in auxin action and that the alteration of auxin distribution corresponded to the curly leaf phenotype. The fact that degeneration of septum cells and subsequent breakage along the stomium was not observed in ptl-D anthers suggests that defective anther dehiscence was the cause for male sterility. Identification and characterization of the gain-of-function mutant ptl-D will improve our understanding of the diverse functions of GT factors during plant development.

Clf swn二重変異体を用いた、Polycombによる胚発生関連因子発現制御機構の解析

clf swn二重変異体を用いた、Polycombによる胚発生関連因子発現制御機構の解析

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish ‘bivalent chromatin marks’ at the silent AGAMOUS locus

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

Cell type-specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase alpha of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity.

In Silico Identification of Co‐transcribed Core Cell Cycle Regulators and Transcription Factors in Arabidopsis

Abstract Regulatory networks involving transcription factors and core cell cycle regulators are expected to play crucial roles in plant growth and development. In this report, we describe the identification of two groups of co‐transcribed core cell cycle regulators and transcription factors via a two‐step in silico screening. The core cell cycle regulators include TARDY ASYNCHRONOUS MEIOSIS (CYCA1;2), CYCB1;1, CYCB2;1, CDKB1;2, and CDKB2;2 while the transcription factors include CURLY LEAF, AINTEGUMENTA, a MYB protein, two Forkhead‐associated domain proteins, and a SCARECROW family protein. Promoter analysis revealed a potential web of cross‐ and self‐regulations among the identified proteins. Because one criterion for screening for these genes is that they are predominantly transcribed in young organs but not in mature organs, these genes are likely to be particularly involved in Arabidopsis organ growth.

14. Roles of CURLY LEAF (CLF) and SWINGER (SWN) in the repression of embryonic trait in Arabidopsis thaliana

14. Roles of CURLY LEAF (CLF) and SWINGER (SWN) in the repression of embryonic trait in Arabidopsis thaliana

The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3.

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27

The plant Polycomb-group (Pc-G) protein CURLY LEAF (CLF) is required to repress targets such as AGAMOUS (AG) and SHOOTMERISTEMLESS (STM). Using chromatin immunoprecipitation, we identify AG and STM as direct targets for CLF and show that they carry a characteristic epigenetic signature of dispersed histone H3 lysine 27 trimethylation (H3K27me3) and localised H3K27me2 methylation. H3K27 methylation is present throughout leaf development and consistent with this, CLF is required persistently to silence AG. However, CLF is not itself an epigenetic mark as it is lost during mitosis. We suggest a model in which Pc-G proteins are recruited to localised regions of targets and then mediate dispersed H3K27me3. Analysis of transgenes carrying AG regulatory sequences confirms that H3K27me3 can spread to novel sequences in a CLF-dependent manner and further shows that H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc-G silencing, and that the loss of silencing caused by transposon insertions at plant Pc-G targets reflects impaired spreading.

An Indole-3-Acetic Acid Carboxyl Methyltransferase RegulatesArabidopsisLeaf Development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.

Transgene‐triggered, epigenetically regulated ectopic expression of a flower homeotic gene pMADS3 in Petunia

pMADS3 is a class C floral homeotic gene of Petunia that is specifically expressed in stamens and carpels of developing flowers. We previously reported that introduction of a part of the pMADS3 genomic sequence silenced endogenous pMADS3 (sil-pMADS3) in transgenic Petunia hybrida. Here we report that introduction of the same sequence triggers ectopic expression of endogenous pMADS3 in sepals, petals and leaves (ect-pMADS3), accompanied by homeotic conversion of the floral organs and altered leaf morphology similar to that of an Arabidopsis curly leaf mutant. The occurrence of the ect-pMADS3 phenotype depended on the presence of pMADS3 intron 2 in the transgenes. Occasionally, sil-pMADS3 and ect-pMADS3 phenotypes somatically interconverted. Some T1 progeny inherited their parent's pMADS3 expression pattern, while others switched from sil-pMADS3 to ect-pMADS3 and vice versa. Both phenotypes occasionally occurred even after the transgenes were segregated away. RT-PCR analyses of ectopically expressed pMADS3 transcripts indicated that two pMADS3 alleles were often differently regulated. Furthermore, reciprocal crosses with untransformed Petunia indicated that pMADS3 alleles other than the one ectopically expressed in T0 plants were sometimes expressed ectopically in T1 plants: the paramutation-like transmission of epigenetic regulation between alleles. We detected in the transformants aberrant transcripts, including sense and antisense pMADS3 intron 2 sequences of heterogeneous molecular sizes, irrespective of the pMADS3 phenotypes. We speculate on possible molecular mechanisms underlying these epigenetic phenomena.