Capturing Environmental Plant Memories in DNA, with a Little Help from Chromatin.

Plants are eukaryotes living mostly immotile in harsh environments. On occasion, it is beneficial for their survival to maintain a transcriptional response to an environmental stress longer than the stress lasts (transcriptional memory) and even to reiterate such a response more quickly or more strongly when the same stress is re-encountered (priming memory). In eukaryotes, transcription takes place in the context of chromatin, the packaging material of DNA. Chromatin regulation is often invoked when it comes to environmental transcriptional and priming memory in plants, but rarely chromatin-based regulation can be accurately assigned to a given aspect of transcription in vivo. The conserved eukaryotic chromatin-modifying system Polycomb/Trithorax can support both long-term stability and flexibility of gene expression in Drosophila. The main principles of Polycomb/Trithorax regulation will be outlined and illustrated with the best-studied case of environmental memory from Arabidopsis. Despite being complex, the Polycomb/Trithorax system relies on experimentally tractable elements in the form of DNA, termed Polycomb/Trithorax Responsive Elements. PREs/TREs are essentially memory DNA elements. Here, relevant information to identify PRE/TRE-like elements in plants is highlighted. Examples of priming memory in plants are discussed in relation to the first two reported putative memory DNA elements. Arguably, similar cases from plants can be conducive in dissecting the contribution of DNA-based from chromatin-based regulation of transcription, when two types of DNA elements are defined: those representing binding sites for the transcription factors determining the environmental response and those controlling memory by regulating chromatin modification dynamics, ultimately maintaining the corresponding transcriptional state.

Epigenetic role for the conserved Fe-S cluster biogenesis protein AtDRE2 in Arabidopsis thaliana.

On fertilization in Arabidopsis thaliana, one maternal gamete, the central cell, forms a placenta-like tissue, the endosperm. The DNA glycosylase DEMETER (DME) excises 5-methylcytosine via the base excision repair pathway in the central cell before fertilization, creating patterns of asymmetric DNA methylation and maternal gene expression across DNA replications in the endosperm lineage (EDL). Active DNA demethylation in the central cell is essential for transcriptional activity in the EDL of a set of genes, including FLOWERING WAGENINGEN (FWA). A DME-binding motif for iron-sulfur (Fe-S) cluster cofactors is indispensable for its catalytic activity. We used an FWA-GFP reporter to find mutants defective in maternal activation of FWA-GFP in the EDL, and isolated an allele of the yeast Dre2/human antiapoptotic factor CIAPIN1 homolog, encoding an enzyme previously implicated in the cytosolic Fe-S biogenesis pathway (CIA), which we named atdre2-2. We found that AtDRE2 acts in the central cell to regulate genes maternally activated in the EDL by DME. Furthermore, the FWA-GFP expression defect in atdre2-2 was partially suppressed genetically by a mutation in the maintenance DNA methyltransferase MET1; the DNA methylation levels at four DME targets increased in atdre2-2 seeds relative to WT. Although atdre2-2 shares zygotic seed defects with CIA mutants, it also uniquely manifests dme phenotypic hallmarks. These results demonstrate a previously unidentified epigenetic function of AtDRE2 that may be separate from the CIA pathway.

Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes.

BACKGROUND: Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. RESULTS: We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). CONCLUSIONS: Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3.

The role of Arabidopsis thaliana NAR1, a cytosolic iron-sulfur cluster assembly component, in gametophytic gene expression and oxidative stress responses in vegetative tissue.

Iron-sulfur proteins have iron-sulfur clusters as a prosthetic group and are responsible for various cellular processes, including general transcriptional regulation, photosynthesis and respiration. The cytosolic iron-sulfur assembly (CIA) pathway of yeast has been shown to be responsible for regulation of iron-sulfur cluster assembly in both the cytosol and the nucleus. However, little is known about the roles of this pathway in multicellular organisms. In a forward genetic screen, we identified an Arabidopsis thaliana mutant with impaired expression of the endosperm-specific gene Flowering Wageningen (FWA). To characterize this mutant, we carried out detailed phenotypic and genetic analyses during reproductive and vegetative development. The mutation affects NAR1, which encodes a homolog of a yeast CIA pathway component. Comparison of embryo development in nar1-3 and other A. thaliana mutants affected in the CIA pathway showed that the embryos aborted at a similar stage, suggesting that this pathway potentially functions in early seed development. Transcriptome analysis of homozygous viable nar1-4 seedlings showed transcriptional repression of a subset of genes involved in 'iron ion transport' and 'response to nitrate'. nar1-4 also exhibited resistance to the herbicide paraquat. Our results indicate that A. thaliana NAR1 has various functions including transcriptional regulation in gametophytes and abiotic stress responses in vegetative tissues.

Transcription-dependence of histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC.

The FLC gene encodes a MADS box repressor of flowering that is the main cause of the late-flowering phenotype of many Arabidopsis ecotypes. Expression of FLC is repressed by vernalization; maintenance of this repression is associated with the deposition of histone 3 K27 trimethylation (H3K27me3) at the FLC locus. However, whether this increased H3K27me3 is a consequence of reduced FLC transcription or the cause of transcriptional repression is not well defined. In this study we investigate the effect of changes in transcription rate on the abundance of H3K27me3 in the FLC gene body, a chromatin region that includes sequences required to maintain FLC repression following vernalization. We show that H3K27me3 is inversely correlated with transcription across the FLC gene body in a range of ecotypes and mutants with different flowering times. We demonstrate that the FLC gene body becomes marked with H3K27me3 in the absence of transcription. When transcription of the gene body is directed by an inducible promoter, H3K27me3 is removed following activation of transcription and H3K27me3 is added after transcription is decreased. The rate of addition of H3K27me3 to the FLC transgene following inactivation of transcription is similar to that observed in the FLC gene body following vernalization. Our data suggest that reduction of FLC transcription during vernalization leads to an increase of H3K27me3 levels in the FLC gene body that in turn maintains FLC repression.

Polycomb proteins regulate the quantitative induction of VERNALIZATION INSENSITIVE 3 in response to low temperatures.

Vernalization, the promotion of flowering in response to low temperatures, is one of the best characterized examples of epigenetic regulation in plants. The promotion of flowering is proportional to the duration of the cold period, but the mechanism by which plants measure time at low temperatures has been a long-standing mystery. We show that the quantitative induction of the first gene in the Arabidopsis vernalization pathway, VERNALIZATION INSENSITIVE 3 (VIN3), is regulated by the components of Polycomb Response Complex 2, which trimethylates histone H3 lysine 27 (H3K27me3). In differentiated animal cells, H3K27me3 is mostly associated with long-term gene repression, whereas, in pluripotent embyonic stem cells, many cell lineage-specific genes are inactive but exist in bivalent chromatin that carries both active (H3K4me3) and repressive (H3K27me3) marks on the same molecule. During differentiation, bivalent domains are generally resolved to an active or silent state. We found that H3K27me3 maintains VIN3 in a repressed state prior to cold exposure; this mark is not removed during VIN3 induction. Instead, active VIN3 is associated with bivalently marked chromatin. The continued presence of H3K27me3 ensures that induction of VIN3 is proportional to the duration of the cold, and that plants require prolonged cold to promote the transition to flowering. The observation that Polycomb proteins control VIN3 activity defines a new role for Polycomb proteins in regulating the rate of gene induction.

FLC: a hidden polycomb response element shows up in silence.

A sizeable fraction of eukaryotic genomes is regulated by Polycomb group (PcG) and trithorax group (trxG) proteins, which play key roles in epigenetic repression and activation, respectively. In Drosophila melanogaster, homeotic genes are well-documented PcG targets; they are known to contain cis-acting elements termed Polycomb response elements (PREs), which bind PcG proteins and satisfy three defined criteria, and also often contain binding sites for the trithorax (trx) protein. However, the presence of PREs, or an alternative mode for PcG/trxG interaction with the genome, has not been well documented outside Drosophila. In Arabidopsis thaliana, PcG/trxG regulation has been studied extensively for the flowering repressor gene FLOWERING LOCUS C (FLC). Here we evaluate how PRE-like activities that reside within the FLC locus may satisfy the defined Drosophila criteria, by analyzing four FLC transcription states. When the FLC locus is not transcribed, the intrinsic PcG recruitment ability of the coding region can be attributed to two redundant cis-acting elements (Modules IIA and IIB). When FLC is highly expressed, trxG recruitment is to a region overlapping the transcription start site (Module I). Exposure to prolonged cold converts the active FLC state into a repressed state that is maintained after the cold period finishes. These two additional transcriptional states also rely on the same three modules for PcG/trxG regulation. We conclude that each of Modules I, IIA and IIB partially fulfills the PRE function criteria, and that together they represent the functional FLC PRE, which differs structurally from canonical PREs in Drosophila.

The Flowering Season-Meter at FLOWERING LOCUS C Across Life Histories in Crucifers.

Many plant species overwinter before they flower. Transition to flowering is aligned to the seasonal transition as a response to the prolonged cold in winter by a process called vernalization. Multiple well-documented vernalization properties in crucifer species with diverse life histories are derived from environmental regulation of a central inhibitor of the flowering gene, Flowering Locus C (FLC). Episode(s) of flowering are prevented during high FLC expression and enabled during low FLC expression. FLC repression outlasts the winter to coincide with spring; this heterochronic aspect is termed "winter memory." In the annual Arabidopsis thaliana, winter memory has long been associated with the highly conserved histone modifiers Polycomb and Trithorax, which have antagonistic roles in transcription. However, there are experimental limitations in determining how dynamic, heterogenous histone modifications within the FLC locus generate the final transcriptional output. Recent theoretical considerations on cell-to-cell variability in gene expression and histone modifications generating bistable states brought support to the hypothesis of chromatin-encoded memory, as with other experimental systems in eukaryotes. Furthermore, these advances unify multiple properties of vernalization, not only the winter memory. Similarly, in the perennial Arabidopsis halleri ssp. gemmifera, recent integration of molecular with mathematical and ecological approaches unifies FLC chromatin features with the all-year-round memory of seasonal temperature. We develop the concept of FLC season-meter to combine existing information from the contrasting annual/perennial and experimental/theoretical sectors into a transitional framework. We highlight simplicity, high conservation, and discrete differences across extreme life histories in crucifers.

Repressive chromatin modification underpins the long-term expression trend of a perennial flowering gene in nature.

Natural environments require organisms to possess robust mechanisms allowing responses to seasonal trends. In Arabidopsis halleri, the flowering regulator AhgFLC shows upregulation and downregulation phases along with long-term past temperature, but the underlying machinery remains elusive. Here, we investigate the seasonal dynamics of histone modifications, H3K27me3 and H3K4me3, at AhgFLC in a natural population. Our advanced modelling and transplant experiments reveal that H3K27me3-mediated chromatin regulation at AhgFLC provides two essential properties. One is the ability to respond to the long-term temperature trends via bidirectional interactions between H3K27me3 and H3K4me3; the other is the ratchet-like character of the AhgFLC system, i.e. reversible in the entire perennial life cycle but irreversible during the upregulation phase. Furthermore, we show that the long-term temperature trends are locally indexed at AhgFLC in the form of histone modifications. Our study provides a more comprehensive understanding of H3K27me3 function at AhgFLC in a complex natural environment.

UBIQUITIN-SPECIFIC PROTEASE 26 is required for seed development and the repression of PHERES1 in Arabidopsis.

The Arabidopsis mutant Atubp26 initiates autonomous endosperm at a frequency of approximately 1% in the absence of fertilization and develops arrested seeds at a frequency of approximately 65% when self-pollinated. These phenotypes are similar to those of the FERTILIZATION INDEPENDENT SEED (FIS) class mutants, mea, fis2, fie, and Atmsi1, which also show development of the central cell into endosperm in the absence of fertilization and arrest of the embryo following fertilization. Atubp26 results from a T-DNA insertion in the UBIQUITIN-SPECIFIC PROTEASE gene AtUBP26, which catalyzes deubiquitination of histone H2B and is required for heterochromatin silencing. The paternal copy of AtUBP26 is able to complement the loss of function of the maternal copy in postfertilization seed development. This contrasts to the fis class mutants where the paternal FIS copy does not rescue aborted seeds. As in the fis class mutants, the Polycomb group (PcG) complex target gene PHERES1 (PHE1) is expressed at higher levels in Atubp26 ovules than in wild type; there is a lower level of H3K27me3 at the PHE1 locus. The phenotypes suggest that AtUBP26 is required for normal seed development and the repression of PHE1.

Short- and long-distance control of root development by LjHAR1 during the juvenile stage of Lotus japonicus.

The autoregulation of nodulation (AON) is a universal mechanism to legumes to control the extent of nodulation via a systemic circuit and if genetically altered, as in the Lotus japonicus har1-1 mutant, leads to hypernodulation and aberrant root development. Increased nodulation of har1-1 is associated with pleiotropic effects both in the absence and presence of the symbiosis. We used two different grafting techniques to investigate the control of the non-symbiotic retarded root growth phenotype of har1-1, and demonstrate that altered root growth in the non-symbiotic condition is controlled by the genotype of both the shoot and the root. Based on these results and on the Gresshoff and Delves [Plant genetic approaches to symbiotic nodulation and nitrogen fixation in legumes. Plant Gene Res 1986;3:159-206] AON model, we propose an advanced working model for control of root development by LjHAR1.

Emerging links between iron-sulfur clusters and 5-methylcytosine base excision repair in plants.

Iron-sulfur (Fe-S) clusters are ancient cofactors present in all kingdoms of life. Both the Fe-S cluster assembly machineries and target apoproteins are distributed across different subcellular compartments. The essential function of Fe-S clusters in nuclear enzymes is particularly difficult to study. The base excision repair (BER) pathway guards the integrity of DNA; enzymes from the DEMETER family of DNA glycosylases in plants are Fe-S cluster-dependent and extend the BER repertowere to excision of 5-methylcytosine (5mC). Recent studies in plants genetically link the majority of proteins from the cytosolic Fe-S cluster biogenesis (CIA) pathway with 5mC BER and DNA repair. This link can now be further explored. First, it opens new possibilities for understanding how Fe-S clusters participate in 5mC BER and related processes. I describe DNA-mediated charge transfer, an Fe-S cluster-based mechanism for locating base lesions with high efficiency, which is used by bacterial DNA glycosylases encoding Fe-S cluster binding domains that are also conserved in the DEMETER family. Second, because detailed analysis of the mutant phenotype of CIA proteins relating to 5mC BER revealed that they formed two groups, we may also gain new insights into both the composition of the Fe-S assembly pathway and the biological contexts of Fe-S proteins.

From the laboratory to the field: assaying histone methylation at FLOWERING LOCUS C in naturally growing Arabidopsis halleri.

Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, "Field ChIP", was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin analysis of AhgFLC in the future to contribute to our understanding of gene regulation in fluctuating natural environments.

Vernalization-repression of Arabidopsis FLC requires promoter sequences but not antisense transcripts.

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts.

Promoter trapping in Lotus japonicus reveals novel root and nodule GUS expression domains.

Agrobacterium-based transformation was used to introduce a promoter-less glucuronidase uidA gene (beta-glucuronidase; GUS) into Lotus japonicus. Transgenic plants were screened for GUS activation at different stages after inoculation with its symbiont, Mesorhizobium loti. Functional GUS fusion frequencies ranged from about 2 to 5% of the total number of transgenic lines. These lines provide excellent histological markers for tissue ontogeny analysis. Some of the activations generated GUS expression patterns that correspond to well-known tissue types, such as lateral root and nodule primordia, root tips and developing nodules (line CHEETAH). Others generated GUS activation associated with predictable but previously unknown (i) tissue types, such as the vascular bundle of the nodule (line VASCO); or (ii) expression domains, such as pericycle, nodule primordia, nodule and flower connective/vascular tissue (line FATA MORGANA) or inner root cortex cells in the vicinity of a curled root hair, nodule primordia and nodule cortex (line TIMPA). Putative members of two gene superfamilies, EH (Esp homolog) and AAA ATPase (ATPase associated with various cellular activities), were located next to the CHEETAH and VASCO insertions, respectively, and a nodulin gene, LjENOD40-2, was located next to the FATA MORGANA insertion. We utilized promoter GUS fusions to investigate the genetic regulation of LjENOD40-2 and FATA MORGANA GUS. The LjENOD40-2 promoter defined a novel expression domain and the FATA MORGANA nodule expression was reiterated by the 2 kb sequence upstream of the T-DNA insertion.

Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase.

Proliferation of legume nodule primordia is controlled by shoot-root signaling known as autoregulation of nodulation (AON). Mutants defective in AON show supernodulation and increased numbers of lateral roots. Here, we demonstrate that AON in soybean is controlled by the receptor-like protein kinase GmNARK (Glycine max nodule autoregulation receptor kinase), similar to Arabidopsis CLAVATA1 (CLV1). Whereas CLV1 functions in a protein complex controlling stem cell proliferation by short-distance signaling in shoot apices, GmNARK expression in the leaf has a major role in long-distance communication with nodule and lateral root primordia.