Role of H1 and DNA methylation in selective regulation of transposable elements during heat stress.

Heat-stressed Arabidopsis plants release heterochromatin-associated transposable element (TE) silencing, yet it is not accompanied by major reductions of epigenetic repressive modifications. In this study, we explored the functional role of histone H1 in repressing heterochromatic TEs in response to heat stress. We generated and analyzed RNA and bisulfite-sequencing data of wild-type and h1 mutant seedlings before and after heat stress. Loss of H1 caused activation of pericentromeric Gypsy elements upon heat treatment, despite these elements remaining highly methylated. By contrast, nonpericentromeric Copia elements became activated concomitantly with loss of DNA methylation. The same Copia elements became activated in heat-treated chromomethylase 2 (cmt2) mutants, indicating that H1 represses Copia elements through maintaining DNA methylation under heat. We discovered that H1 is required for TE repression in response to heat stress, but its functional role differs depending on TE location. Strikingly, H1-deficient plants treated with the DNA methyltransferase inhibitor zebularine were highly tolerant to heat stress, suggesting that both H1 and DNA methylation redundantly suppress the plant response to heat stress.

Dark-Induced Senescence Causes Localized Changes in DNA Methylation.

Senescence occurs in a programmed manner to dismantle the vegetative tissues and redirect nutrients towards metabolic pathways supporting reproductive success. External factors can trigger the senescence program as an adaptive strategy, indicating that this terminal program is controlled at different levels. It has been proposed that epigenetic factors accompany the reprogramming of the senescent genome; however, the mechanism and extent of this reprogramming remain unknown. Using bisulphite conversion followed by sequencing, we assessed changes in the methylome of senescent Arabidopsis (Arabidopsis thaliana) leaves induced by darkness and monitored their effect on gene and transposable element (TE) expression with transcriptome sequencing. Upon dark-induced senescence, genes controlling chromatin silencing were collectively down-regulated. As a consequence, the silencing of TEs was impaired, causing in particular young TEs to become preferentially reactivated. In parallel, heterochromatin at chromocenters was decondensed. Despite the disruption of the chromatin maintenance network, the global DNA methylation landscape remained highly stable, with localized changes mainly restricted to CHH methylation. Together, our data show that the terminal stage of plant life is accompanied by global changes in chromatin structure but only localized changes in DNA methylation, adding another example of the dynamics of DNA methylation during plant development.

Salicylic acid interferes with GFP fluorescence in vivo.

Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.

Low Temperature Affects Stem Cell Maintenance in Brassica oleracea Seedlings.

Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.

Natural CMT2 variation is associated with genome-wide methylation changes and temperature seasonality.

As Arabidopsis thaliana has colonized a wide range of habitats across the world it is an attractive model for studying the genetic mechanisms underlying environmental adaptation. Here, we used public data from two collections of A. thaliana accessions to associate genetic variability at individual loci with differences in climates at the sampling sites. We use a novel method to screen the genome for plastic alleles that tolerate a broader climate range than the major allele. This approach reduces confounding with population structure and increases power compared to standard genome-wide association methods. Sixteen novel loci were found, including an association between Chromomethylase 2 (CMT2) and temperature seasonality where the genome-wide CHH methylation was different for the group of accessions carrying the plastic allele. Cmt2 mutants were shown to be more tolerant to heat-stress, suggesting genetic regulation of epigenetic modifications as a likely mechanism underlying natural adaptation to variable temperatures, potentially through differential allelic plasticity to temperature-stress.