Accumulation of the coumarin scopolin under abiotic stress conditions is mediated by the Arabidopsis thaliana THO/TREX complex.

Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function.

Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 µm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

DNA methylation maintenance consolidates RNA-directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana.

In plants, 24 nucleotide short interfering RNAs serve as a signal to direct cytosine methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter function, this RNA-directed DNA methylation may result in transcriptional gene silencing. In a genetic screen for factors involved in RNA-directed transcriptional silencing of a ProNOS-NPTII reporter transgene in Arabidopsis thaliana, we captured alleles of DOMAINS REARRANGED METHYLTRANSFERASE 2, the gene encoding the DNA methyltransferase that is mainly responsible for de novo DNA methylation in the context of RNA-directed DNA methylation. Interestingly, methylation of the reporter gene ProNOS was not completely erased in these mutants, but persisted in the symmetric CG context, indicating that RNA-directed DNA methylation had been consolidated by DNA methylation maintenance. Taking advantage of the segregation of the transgenes giving rise to ProNOS short interfering RNAs and carrying the ProNOS-NPTII reporter in our experimental system, we found that ProNOS DNA methylation maintenance was first evident after two generations of ongoing RNA-directed DNA methylation, and then increased in extent with further generations. As ProNOS DNA methylation had already reached its final level in the first generation of RNA-directed DNA methylation, our findings suggest that establishment of DNA methylation at a particular region may be divided into distinct stages. An initial phase of efficient, but still fully reversible, de novo DNA methylation and transcriptional gene silencing is followed by transition to efficient maintenance of cytosine methylation in a symmetric sequence context accompanied by persistence of gene silencing.

Additive inheritance of histone modifications in Arabidopsis thaliana intra-specific hybrids.

Plant genomes are earmarked with defined patterns of chromatin marks. Little is known about the stability of these epigenomes when related, but distinct genomes are brought together by intra-species hybridization. Arabidopsis thaliana accessions and their reciprocal hybrids were used as a model system to investigate the dynamics of histone modification patterns. The genome-wide distribution of histone modifications H3K4me2 and H3K27me3 in the inbred parental accessions Col-0, C24 and Cvi and their hybrid offspring was compared by chromatin immunoprecipitation in combination with genome tiling array hybridization. The analysis revealed that, in addition to DNA sequence polymorphisms, chromatin modification variations exist among accessions of A. thaliana. The range of these variations was higher for H3K27me3 (typically a repressive mark) than for H3K4me2 (typically an active mark). H3K4me2 and H3K27me3 were rather stable in response to intra-species hybridization, with mainly additive inheritance in hybrid offspring. In conclusion, intra-species hybridization does not result in gross changes to chromatin modifications.

Influence of cryopreservation on the cytosine methylation state of potato genomic NDA.

Shoot tips of Solanum tuberosum (Désirée) were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9 % of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6 % (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.

Predicting Hybrid Performances for Quality Traits through Genomic-Assisted Approaches in Central European Wheat.

Bread-making quality traits are central targets for wheat breeding. The objectives of our study were to (1) examine the presence of major effect QTLs for quality traits in a Central European elite wheat population, (2) explore the optimal strategy for predicting the hybrid performance for wheat quality traits, and (3) investigate the effects of marker density and the composition and size of the training population on the accuracy of prediction of hybrid performance. In total 135 inbred lines of Central European bread wheat (Triticum aestivum L.) and 1,604 hybrids derived from them were evaluated for seven quality traits in up to six environments. The 135 parental lines were genotyped using a 90k single-nucleotide polymorphism array. Genome-wide association mapping initially suggested presence of several quantitative trait loci (QTLs), but cross-validation rather indicated the absence of major effect QTLs for all quality traits except of 1000-kernel weight. Genomic selection substantially outperformed marker-assisted selection in predicting hybrid performance. A resampling study revealed that increasing the effective population size in the estimation set of hybrids is relevant to boost the accuracy of prediction for an unrelated test population.

Biparental resequencing coupled with SNP genotyping of a segregating population offers insights into the landscape of recombination and fixed genomic regions in elite soybean.

Identification of genes underlying agronomic traits is dependent on the segregation of quantitative trait loci (QTL). A popular hypothesis is that elite lines are becoming increasingly similar to each other, resulting in large genomic regions with fixed genes. Here, we resequenced two parental modern elite soybean lines [ZhongHuang13 (ZH) and ZhongPin03-5373 (ZP)] and discovered 794,876 SNPs with reference to the published Williams82 genome. SNPs were distributed unevenly across the chromosomes, with 87.1% of SNPs clustering in 4.9% of the soybean reference genome. Most of the regions with a high density of SNP polymorphisms were located in the chromosome arms. Moreover, seven large regions that were highly similar between parental lines were identified. A GoldenGate SNP genotyping array was designed using 384 SNPs and the 254 recombinant inbred lines (F8) derived from the cross of ZP × ZH were genotyped. We constructed a genetic linkage map using a total of 485 molecular markers, including 313 SNPs from the array, 167 simple sequence repeats (SSRs), 4 expressed sequence tag-derived SSRs, and 1 insertion/deletion marker. The total length of the genetic map was 2594.34 cM, with an average marker spacing of 5.58 cM. Comparing physical and genetic distances, we found 20 hotspot and 14 coldspot regions of recombination. Our results suggest that the technology of resequencing of parental lines coupled with high-throughput SNP genotyping could efficiently bridge the genotyping gap and provide deep insights into the landscape of recombination and fixed genomic regions in biparental segregating populations of soybean with implications for fine mapping of QTL.

Novel microRNAs and microsatellite-like small RNAs in sexual and apomictic Boechera species.

Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets.

Engineered plant minichromosomes.

Minichromosomes offer an enormous potential for plant breeding and biotechnology, because they may simultaneously transfer and stably express multiple genes. Segregating independently of their host chromosomes, they provide a platform for accelerating plant breeding. Minichromosomes can be established from cloned components in vivo (bottom up) or via engineering of natural chromosomes (top down). When they possess functional centromeres and telomeres, they should be stably inherited, but their meiotic transmission rate is below that of endogenous chromosomes. To achieve the customized generation and control the regular transmission of minichromosomes are important challenges for applied research in chromosome biology. Here, construction and biology of plant minichromosomes are compared with data available for yeast and animal systems.

Local DNA features affect RNA-directed transcriptional gene silencing and DNA methylation.

Transcription of a nopaline synthase promoter (pNOS) inverted repeat provides an RNA signal that can trigger transcriptional gene silencing and methylation of pNOS promoters in trans. The degree of silencing is influenced by the local DNA features close to the target promoter integration sites. Among 26 transgenic Arabidopsis thaliana lines harbouring single copies of a T-DNA including a pNOS-NPTII reporter gene at different chromosomal loci, NPTII RNA levels showed limited variation. When challenged by the silencer transgene providing the pNOS RNA signal, reduction of the NPTII RNA levels in the F(1) generation varied by more than 100-fold, ranging from no reduction to reduction to <1% of the non-silenced level. Silencing was generally correlated with proportional DNA methylation in the pNOS region, except for one target transgene showing substantial DNA methylation without adequate silencing. Silencing was progressive through generations. Differences in the degree of silencing among the target transgenes were transmitted at least to the F(3) generation, and seemed to be influenced by transgene-flanking sequences. Apparently, close-by repeats promoted, whereas close-by functional genes diminished, the response to the silencing signal.

Size and number of tandem repeat arrays can determine somatic homologous pairing of transgene loci mediated by epigenetic modifications in Arabidopsis thaliana nuclei.

The chromosomal arrangement of different transgenic repeat arrays inserted at various chromosomal positions was tested by FISH in Arabidopsis 2C leaf and root nuclei. Large lacO ( approximately 10 kb) but not tetO (4.8 kb) or small lacO ( approximately 2 kb) arrays were, in general, more often spatially associated with heterochromatic chromocenters (CC) than flanking regions (that either overlap the array insert position or are between 5 and 163 kb apart from the insert site). Allelic and ectopic pairing frequencies of lacO arrays were significantly increased only in nuclei of lines with two large lacO arrays inserted at different positions on the same chromosome arm. Within the same lines, root nuclei showed a significantly lower increase of pairing frequencies at the insert position compared to leaf nuclei but still a higher frequency than in the wild-type situation. Thus, the frequencies of homologous pairing and association with heterochromatin of transgenic repeats may differ with the construct, the chromosomal insertion position, the cell type and with the number and repetitiveness of inserts. Strong CpG methylation is correlated with a high frequency of homologous pairing at large repeat array loci in somatic cells but has no impact on their association with CCs. These results show that single low-copy arrays apparently do not alter interphase chromatin architecture and are more suitable for chromatin tagging than multiple high copy arrays.