The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Mutations in GTP cyclohydrolase I (GCHI) are found in 50 to 60% of cases with dopa-responsive dystonia (DRD). Heterozygous GCHI exon deletions, undetectable by sequencing, have recently been described in three DRD families. We tested 23 individuals with DRD for the different mutation types by conventional and quantitative PCR analyses and found mutations, including two large exon deletions, in 87%. The authors attribute this high mutation rate to rigorous inclusion criteria and comprehensive mutational analysis.

Background: Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. Result: We subjected A. thaliana plants to cold acclimation conditions (4°C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cisand trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. Conclusion: In A. thaliana, miRNAs and sRNAs derived from cisand trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.

Chloroplast perturbations activate retrograde signalling pathways, causing dynamic changes of gene expression. Besides transcriptional control of gene expression, different classes of small non-coding RNAs (sRNAs) act in gene expression control, but comprehensive analyses regarding their role in retrograde signalling are lacking. We performed sRNA profiling in response to norflurazon (NF), which provokes retrograde signals, in Arabidopsis thaliana wild type (WT) and the two retrograde signalling mutants gun1 and gun5. The RNA samples were also used for mRNA and long non-coding RNA profiling to link altered sRNA levels to changes in the expression of their cognate target RNAs. We identified 122 sRNAs from all known sRNA classes that were responsive to NF in the WT. Strikingly, 142 and 213 sRNAs were found to be differentially regulated in both mutants, indicating a retrograde control of these sRNAs. Concomitant with the changes in sRNA expression, we detected about 1500 differentially expressed mRNAs in the NF-treated WT and around 900 and 1400 mRNAs that were differentially regulated in the gun1 and gun5 mutants, with a high proportion (~30%) of genes encoding plastid proteins. Furthermore, around 20% of predicted miRNA targets code for plastid-localised proteins. Among the sRNA-target pairs, we identified pairs with an anticorrelated expression as well pairs showing other expressional relations, pointing to a role of sRNAs in balancing transcriptional changes upon retrograde signals. Based on the comprehensive changes in sRNA expression, we assume a considerable impact of sRNAs in retrograde-dependent transcriptional changes to adjust plastidic and nuclear gene expression.

The biological significance of non-coding RNAs (ncRNAs) has been firmly established to be important for the regulation of genes involved in stress acclimation. Light plays an important role for the growth of plants providing the energy for photosynthesis; however, excessive light conditions can also cause substantial defects. Small RNAs (sRNAs) are a class of non-coding RNAs that regulate transcript levels of protein-coding genes and mediate epigenetic silencing. Next generation sequencing facilitates the identification of small non-coding RNA classes such as miRNAs (microRNAs) and small-interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs), but changes in the ncRNA transcriptome in response to high light are poorly understood. We subjected Arabidopsis plants to high light conditions and performed a temporal in-depth study of the transcriptome data after 3 h, 6 h, and 2 days of high light treatment. We identified a large number of high light responsive miRNAs and sRNAs derived from NAT gene pairs, lncRNAs and TAS transcripts. We performed target predictions for differentially expressed miRNAs and correlated their expression levels through mRNA sequencing data. GO analysis of the targets revealed an overrepresentation of genes involved in transcriptional regulation. In A. thaliana, sRNA-mediated regulation of gene expression in response to high light treatment is mainly carried out by miRNAs and sRNAs derived from NAT gene pairs, and from lncRNAs. This study provides a deeper understanding of sRNA-dependent regulatory networks in high light acclimation.

staining material, heterochromatin, that can be differentiated cytologically from the surrounding lightly staining chromosomal regions (Cavalli and Paro, 1998; Holm-The Cold Spring Harbor Laboratory, Washington University Genome Sequencing Center, and PE Biosystems Arabidopsis quist et al., 1998). In Drosophila and maize, heterochro-Sequencing Consortium* matic regions have been shown to suppress recombination, to deter transposon insertion, and to influence the expression of genes found nearby (Cook and Karpen, Summary 1994; Henikoff, 1998; Dimitri and Junakovic, 1999). In Drosophila, the composition of heterochromatin has Heterochromatin, constitutively condensed chromobeen extensively investigated in minichromosomes desomal material, is widespread among eukaryotes but rived from chromosome 4. Sample sequencing and FISH incompletely characterized at the nucleotide level. We (fluorescent in situ hybridization) have revealed that have sequenced and analyzed 2.1 megabases (Mb) much of the minichromosome comprised arrays of short of Arabidopsis thaliana chromosome 4 that includes satellite repeats, but pulsed-field gel analysis revealed 0.5-0.7 Mb of isolated heterochromatin that resembles islands of low complexity DNA (Le et al., 1995). Some the chromosomal knobs described by Barbara McClinof these islands contained intact retrotransposable eletock in maize. This isolated region has a low density ments, but none were found to be unique to centromeric regions (Sun et al., 1997). In maize, heterochromatin of expressed genes, low levels of recombination and has been extensively investigated since chromosome a low incidence of genetrap insertion. Satellite repeats staining procedures were sensitive enough to detect were absent, but tandem arrays of long repeats and them (McClintock, 1929, 1930; McClintock et al., 1981). many transposons were found. Methylation of these Heterochromatin is found in large pericentromeric dosequences was dependent on chromatin remodeling. mains up to 100 megabases (Mb) in size as well as at Clustered repeats were associated with condensed nucleolar organizers. Smaller islands of heterochromachromosomal domains elsewhere. The complete setin, known as knobs and chromomeres, are distributed quence of a heterochromatic island provides an opalong the chromosome arms, though they are often teloportunity to study sequence determinants of chromomeric. Knobs occur at different positions in different some condensation. strains of maize and are stably polymorphic, allowing their extensive use as cytogenetic landmarks (Richards

Chloroplast perturbations activate retrograde signalling pathways causing dynamic changes of gene expression. Besides transcriptional control of gene expression different classes of small non-coding RNAs (sRNAs) act in gene expression control, but comprehensive analyses regarding their role in retrograde signalling is lacking. We performed sRNA profiling in response to norflurazon (NF) that provokes retrograde signals in A. thaliana wild type and the two retrograde signalling mutants gun1 and gun5. The RNA samples were also used for mRNA and long non-coding RNA (lncRNA) profiling to link altered sRNA levels to changes of their cognate target RNAs. We identified 122 sRNAs from all known sRNA classes that were responsive to NF in wild type. Strikingly, 140 and 213 sRNAs were found to be differentially regulated in both mutants indicating a retrograde control of these sRNAs. Concomitant with the changes in sRNA expression we detected about 1500 differentially expressed mRNAs in the NF treated wild type and around 900 and 1400 mRNAs that were differentially regulated in the gun1 and gun5 mutant with a high proportion (~30%) of genes encoding plastid proteins. Furthermore, around 20% of predicted miRNA targets code for plastid localised proteins. The analyses of sRNA-target pairs identified pairs with an anticorrelated expression as well pairs showing other expressional relations pointing to a role of sRNAs in balancing transcriptional changes upon retrograde signals. Based on the comprehensive changes in sRNA expression we assume a considerable impact of sRNAs in retrograde-dependent transcriptional changes to adjust plastidic and nuclear gene expression. Significance statementPerturbations of plastid functions trigger retrograde signalling to adjust plastidic and nuclear gene expression, however, the role of small non-coding RNAs acting as regulators in these pathways is not well understood. We analysed small non-coding RNA expression in response to retrograde signals in A. thaliana wild type and two retrograde signalling mutants and identified members of all known small non-coding RNA classes pointing to a functional role of these RNA classes in retrograde pathways.