Large-scale chromatin de-compaction induced by low light is not accompanied by nucleosomal displacement

Arabidopsis thaliana is widely used as a model to study chromatin compaction dynamics during development and in response to the environment. Signals such as prolonged heat treatment, low light and pathogen infestation are known to induce large-scale de-condensation of nuclear chromatin. Here we demonstrate that the response to different environments varies at the nucleosomal level. Our results show that in contrast to previous reports on heat and biotic infestation, low light intensity signaling does not alter nucleosomal occupancy, despite the marked effects of low light on global chromatin compaction.Key words: Arabidopsis, chromatin, nucleosomes, MNase IThanks to its relatively simple chromatin organization, Arabidopsis thaliana became the model of choice to study dynamics in nuclear chromatin compaction in plants.^1–3 At the microscopic level, highly condensed ‘heterochromatic’ domains (chromocenters), containing compact DNA (mainly repetitive sequences), and less condensed gene-rich ‘euchromatic’ domains can be distinguished upon staining with DAPI (4′,6-diamidino-2-phenylindole). This division however, is not static and compaction changes throughout development (reviewed in ref. ⁴). Chromatin for example de-condensates prior to flowering⁵ and increases with cell differentiation during leaf maturation³ and seedling establishment.⁶ Vice versa, artificially induced cell de-differentiation during protoplastization, results in loosening of compact chromatin.^7,8 Chromatin compaction is also influenced by various environmental signals. These include infestation by pathogenic microorganisms such as Pseudomonas syringae, light and heat signals.^9–11In our recent paper, published in Plant Physiology,¹² we demonstrate that a ∼90% decrease in light intensity (low light) induces a reversible reduction in global chromatin compaction. In addition, also specifically lowering the blue-light wavelengths in the spectrum, or lowering the red-to-far red (R/Fr) ratio induced a significant reduced compaction of the nuclear chromatin. This is interesting from a functional perspective because (1) these are the relevant signals perceived by plants in natural shade conditions occurring in dense-vegetations and (2) because these wavelengths are specifically detected by the light-sensitive photoreceptor proteins. Previously, we demonstrated that the R/Fr-photoreceptor Phytochrome-B (PhyB) is a positive regulator of chromatin compaction in standard light conditions.¹⁰ We now showed that PhyB also controls low light-induced chromatin organization, but that its effect depend on the genetic background of the phyb mutant under study. Likely, PhyB exerts its effects on light-mediated chromatin compaction via stabilization of CRYPTOCHROME 2 (CRY2) protein. This chromatin-associated blue light photoreceptor is a general positive regulator of low light-induced chromatin de-compaction and in addition controls chromatin compaction during floral transition.⁵In addition, we demonstrated that global chromatin de-compaction during floral transition and low light treatment also occurs in euchromatic domains.^5,12 To study possible chromatin changes at the nucleosomal level, we performed Micrococcal Nuclease I (MNase I) analysis. No differences were observed in the nucleosomal occupancy between standard and low light conditions in DNA gels or Southern blots hybridized with different probes for repeated sequences associated to heterochromatin, and dispersed upon low light treatment (Fig. 1). This suggests that the large-scale heterochromatin (de)compaction response observed at the microscopic level under low light conditions is not necessarily accompanied by nucleosomal displacement. These results are in line with the de-condensation conditions induced by protoplastization, where no changes in H3K9Me2 or in DNA methylation (5-mC) levels were found.⁷ However, these results are in contrast to the results of Pecinka and colleagues,¹¹ who demonstrated that prolonged heat stress results in heterochromatin de-condensation and loss of nucleosomes. Moreover, it is in contrast with Pavet and co-workers,⁹ who found reduced 5-mC levels upon infection with P. syringae. Although the results of Pecinka and colleaugues¹¹ were obtained by real-time PCR which may be more sensitive than our Southern blots, we conclude that the response of plants to their environment at the chromatin compaction level may be tailored to the specific signal it is confronted with and that this probably can be dissected at the nucleosomal level.Open in a separate windowFigure 1MNase I analysis of low light treated plants. Southern blots with 3 different probes hybridized to DNA from Col-0 plants cultured under standard (200 µmol m⁻² s⁻¹; control) and low light (15 µmol m⁻² s⁻¹) conditions. For each part, the first two lanes represent control DNA samples (no MNase I), followed by lanes with increasing MNase I concentrations (0.02, 0.1, 0.75 and 3 units MNase I). (A) 5S rDNA probe, (B) 45S rDNA probe, (C) pAl1 probe (180 bp centromeric repeat). M = molecular weight marker.