Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Epigenetic nature of Arabidopsis thaliana telomeres

The epigenetic features of defined chromosomal domains condition their biochemical and functional properties. Therefore, there is considerable interest in studying the epigenetic marks present at relevant chromosomal loci. Telomeric regions, which include telomeres and subtelomeres, have been traditionally considered heterochromatic. However, whereas the heterochromatic nature of subtelomeres has been widely accepted, the epigenetic status of telomeres remains controversial. Here, we studied the epigenetic features of Arabidopsis (Arabidopsis thaliana) telomeres by analyzing multiple genome-wide ChIP-seq experiments. Our analyses revealed that Arabidopsis telomeres are not significantly enriched either in euchromatic marks like H3K4me2, H3K9ac, and H3K27me3 or in heterochromatic marks such as H3K27me1 and H3K9me2. Thus, telomeric regions in Arabidopsis have a bimodal chromatin organization with telomeres lacking significant levels of canonical euchromatic and heterochromatic marks followed by heterochromatic subtelomeres. Since heterochromatin is known to influence telomere function, the heterochromatic modifications present at Arabidopsis subtelomeres could play a relevant role in telomere biology.

Telomeric regions in Arabidopsis have a bimodal chromatin with telomeres lacking substantial levels of canonical euchromatic and heterochromatic marks followed by heterochromatic subtelomeres.     

Epigenetic and epigenomic variation in Arabidopsis thaliana

Arabidopsis thaliana (Arabidopsis) is ideally suited for studies of natural phenotypic variation. This species has also provided an unparalleled experimental system to explore the mechanistic link between genetic and epigenetic variation, especially with regard to cytosine methylation. Using high-throughput sequencing methods, genotype to epigenotype to phenotype observations can now be extended to plant populations. We review the evidence for induced and spontaneous epigenetic variants that have been identified in Arabidopsis in the laboratory and discuss how these experimental observations could explain existing variation in the wild.     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.     

Lipopolysaccharide mobility in leaf tissue of Arabidopsis thaliana

Bacterial lipopolysaccharides (LPS) are triggers of defence responses in plants, and induce local as well as systemic acquired resistance. Arabidopsis thaliana plants pretreated with LPS show an increased resistance to the virulent bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000. To investigate the mobilization and transport of LPS in Arabidopsis leaves, fluorescently labelled LPS (Alexa Fluor® 488 conjugate) from Salmonella minnesota was used. Leaves were pressure infiltrated with fluorescein‐labelled LPS and fluorescence microscopy was used to follow the movement and localization of LPS as a function of time. The observation of leaves 1 h after supplementation with fluorescein‐labelled LPS revealed a fluorescent signal in the intercellular space. Capillary zone electrophoresis was used for the detection and analysis of the labelled LPS in directly treated leaves and systemic leaves. In addition, gel electrophoresis was used to confirm LPS mobilization. The results indicated that LPS mobilization/translocation occurs through the xylem from local, treated leaves to systemic, untreated leaves. Consequently, care should be taken when ascribing the observed biochemical responses and induced resistance from LPS perception as being uniquely local or systemic, as these responses might overlap because of the mobility of LPS in the plant vascular system.     

Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana

Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.     

Epigenetic variation contributes to environmental adaptation of Arabidopsis thaliana

Epigenetic variation is frequently observed in plants and direct relationships between differences in DNA methylation and phenotypic responses to changing environments have often been described. The identification of contributing genetic loci, however, was until recently hampered by the lack of suitable genome wide mapping resources that specifically segregate for epigenetic marks. The development of epi-RIL populations in the model species Arabidopsis thaliana has alleviated this obstacle, enabling the accurate genetic analysis of epigenetic variation. Comprehensive morphological phenotyping of a ddm1 derived epi-RIL population in different environments and subsequent epi-QTL mapping revealed a high number of epi-QTLs and pleiotropic effects of several DMRs on numerous traits. For a number of these epi-QTLs epistatic interactions could be observed, further adding to the complexity of epigenetic regulation. Moreover, linkage to epigenetic marks indicated a specific role for DNA-methylation variation, rather than TE transposition, in plastic responses to changing environments. These findings provide supportive evidence for a role of epigenetic regulation in evolutionary and adaptive processes.     

Epigenetic Basis of Morphological Variation and Phenotypic Plasticity in Arabidopsis thaliana

Epigenetics is receiving growing attention in the plant science community. Epigenetic modifications are thought to play a particularly important role in fluctuating environments. It is hypothesized that epigenetics contributes to plant phenotypic plasticity because epigenetic modifications, in contrast to DNA sequence variation, are more likely to be reversible. The population of decrease in DNA methylation 1-2 (ddm1-2)-derived epigenetic recombinant inbred lines (epiRILs) in Arabidopsis thaliana is well suited for studying this hypothesis, as DNA methylation differences are maximized and DNA sequence variation is minimized. Here, we report on the extensive heritable epigenetic variation in plant growth and morphology in neutral and saline conditions detected among the epiRILs. Plant performance, in terms of branching and leaf area, was both reduced and enhanced by different quantitative trait loci (QTLs) in the ddm1-2 inherited epigenotypes. The variation in plasticity associated significantly with certain genomic regions in which the ddm1-2 inherited epigenotypes caused an increased sensitivity to environmental changes, probably due to impaired genetic regulation in the epiRILs. Many of the QTLs for morphology and plasticity overlapped, suggesting major pleiotropic effects. These findings indicate that epigenetics contributes substantially to variation in plant growth, morphology, and plasticity, especially under stress conditions.     

A high-copy-number CACTA family transposon in temperate grasses and cereals

A lineage of CACTA family transposons has been identified in temperate grasses and cereals, and a full-length representative of the subfamily from Lolium perenne has been sequenced. Both the size and internal organization of the L. perenne element are typical of other CACTA family elements but its high copy number and strong conservation are unexpected. Comparison with homologs in other species suggests that this lineage has adopted a distinct and novel evolutionary strategy, which has allowed it to maintain its presence in genomes over long periods of time.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Genetic control of petiole length in Arabidopsis thaliana

Shade-avoidance syndrome is characterized by the formation of elongated petioles and unexpanded leaf blades under low-intensity light, but the genetic basis for these responses is unknown. In this study, two-dimensional mutational analysis revealed that the gene for phytochrome B, PHYB, had opposing effects in the leaf petioles and leaf blades of Arabidopsis, while the ROT3, ACL2, and GAI genes influenced the length of leaf petioles more significantly than the length of leaf blades. Anatomical analysis revealed that the PHYB and ACL2 genes control the length of leaf petioles exclusively via control of the length of individual cells, while the GAI, GA1 and ROT3 genes appeared to control both the elongation and proliferation of petiole cells, in particular, under strong light. By contrast, both the size and the number of cells were affected by the mutations examined in leaf blades. The differential control of leaf petiole length and leaf blade expansion is discussed.     

Brassinolide may control aquaporin activities in Arabidopsis thaliana

 It is usually assumed that aquaporins present in the cellular membranes could be an important route in the control of water flux in plants, but evidence for this hypothesis is scarce. In this paper, we report measurements of the osmotic permeability (P _os ) of protoplasts isolated from hypocotyls of wild-type and mutant Arabidopsis thaliana (L.) Heynh. Mutants were affected in their growth and exhibited different sensitivities to the phytohormone, brassinolide. For the two mutants studied (cpd: constitutive photomorphogenesis and dwarfism; bri1: brassinosteroid insensitive), hypocotyl length was correlated to P _os for the protoplasts. Under experimental conditions where hypocotyl growth had ceased, restoration of root, hypocotyl and petiole growth by brassinolide was correlated with an increase in P _os of the hypocotyl protoplasts. We consider that the increase in P _os of the hypocotyl cells was needed because these cells were part of the transcellular water pathway of the plant. This is the first time, to our knowledge, that brassinolide has been shown to be involved in the modification of the water-transport properties of cell membranes. Our results also emphasize the importance of aquaporins and the transcellular pathway in water transport under normal growth conditions. Received: 15 January 2000 / Accepted: 18 May 2000