Distinct guard cell-specific remodeling of chromatin accessibility during abscisic acid- and CO2-dependent stomatal regulation.

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2 (carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type-specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell-specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO2 had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance.

Distinct guard cell specific remodeling of chromatin accessibility during abscisic acid and CO2 dependent stomatal regulation.

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures thereby regulating gas exchange. Chromatin structure controls transcription factor access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remain unknown. Here we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2, regulate guard cell chromatin during stomatal movements. Our cell type specific analyses uncover patterns of chromatin accessibility specific to guard cells and define novel cis-regulatory sequences supporting guard cell specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell-type specificity. DNA motif analyses uncover binding sites for distinct transcription factors enriched in ABA-induced and ABA-repressed chromatin. We identify the ABF/AREB bZIP-type transcription factors that are required for ABA-triggered chromatin opening in guard cells and implicate the inhibition of a set of bHLH-type transcription factors in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance.

Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila.

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. Here, we show that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a "community effect" counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. We propose a model in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins.

Plant hormone regulation of abiotic stress responses.

Plant hormones are signalling compounds that regulate crucial aspects of growth, development and environmental stress responses. Abiotic stresses, such as drought, salinity, heat, cold and flooding, have profound effects on plant growth and survival. Adaptation and tolerance to such stresses require sophisticated sensing, signalling and stress response mechanisms. In this Review, we discuss recent advances in understanding how diverse plant hormones control abiotic stress responses in plants and highlight points of hormonal crosstalk during abiotic stress signalling. Control mechanisms and stress responses mediated by plant hormones including abscisic acid, auxin, brassinosteroids, cytokinins, ethylene and gibberellins are discussed. We discuss new insights into osmotic stress sensing and signalling mechanisms, hormonal control of gene regulation and plant development during stress, hormone-regulated submergence tolerance and stomatal movements. We further explore how innovative imaging approaches are providing insights into single-cell and tissue hormone dynamics. Understanding stress tolerance mechanisms opens new opportunities for agricultural applications.

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila.

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/trimethylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo, the heterochromatin lacks these modifications, which appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (MBT). Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap, we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase but then dissociates in mitosis, and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition.

In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase-promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.

Timing the Drosophila Mid-Blastula Transition: A Cell Cycle-Centered View.

At the mid-blastula transition (MBT), externally developing embryos refocus from increasing cell number to elaboration of the body plan. Studies in Drosophila reveal a sequence of changes in regulators of Cyclin:Cdk1 that increasingly restricts the activity of this cell cycle kinase to slow cell cycles during early embryogenesis. By reviewing these events, we provide an outline of the mechanisms slowing the cell cycle at and around the time of MBT. The perspectives developed should provide a guiding paradigm for the study of other MBT changes as the embryo transits from maternal control to a regulatory program centered on the expression of zygotic genes.

Acetylome profiling reveals overlap in the regulation of diverse processes by sirtuins, gcn5, and esa1.

Although histone acetylation and deacetylation machineries (HATs and HDACs) regulate important aspects of cell function by targeting histone tails, recent work highlights that non-histone protein acetylation is also pervasive in eukaryotes. Here, we use quantitative mass-spectrometry to define acetylations targeted by the sirtuin family, previously implicated in the regulation of non-histone protein acetylation. To identify HATs that promote acetylation of these sites, we also performed this analysis in gcn5 (SAGA) and esa1 (NuA4) mutants. We observed strong sequence specificity for the sirtuins and for each of these HATs. Although the Gcn5 and Esa1 consensus sequences are entirely distinct, the sirtuin consensus overlaps almost entirely with that of Gcn5, suggesting a strong coordination between these two regulatory enzymes. Furthermore, by examining global acetylation in an ada2 mutant, which dissociates Gcn5 from the SAGA complex, we found that a subset of Gcn5 targets did not depend on an intact SAGA complex for targeting. Our work provides a framework for understanding how HAT and HDAC enzymes collaborate to regulate critical cellular processes related to growth and division.

Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins.

We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin-associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using ligase traps of eight different F box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously unreported, substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F box partner, but not other ligase traps. Interestingly, the four most abundant candidate substrates identified for the F box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation.

Gcn5 and sirtuins regulate acetylation of the ribosomal protein transcription factor Ifh1.

BACKGROUND: In eukaryotes, ribosome biosynthesis involves the coordination of ribosomal RNA and ribosomal protein (RP) production. In S. cerevisiae, the regulation of ribosome biosynthesis occurs largely at the level of transcription. The transcription factor Ifh1 binds at RP genes and promotes their transcription when growth conditions are favorable. Although Ifh1 recruitment to RP genes has been characterized, little is known about the regulation of promoter-bound Ifh1. RESULTS: We used a novel whole-cell-extract screening approach to identify Spt7, a member of the SAGA transcription complex, and the RP transactivator Ifh1 as highly acetylated nonhistone species. We report that Ifh1 is modified by acetylation specifically in an N-terminal domain. These acetylations require the Gcn5 histone acetyltransferase and are reversed by the sirtuin deacetylases Hst1 and Sir2. Ifh1 acetylation is regulated by rapamycin treatment and stress and limits the ability of Ifh1 to act as a transactivator at RP genes. CONCLUSIONS: Our data suggest a novel mechanism of regulation whereby Gcn5 functions to titrate the activity of Ifh1 following its recruitment to RP promoters to provide more than an all-or-nothing mode of transcriptional regulation. We provide insights into how the action of histone acetylation machineries converges with nutrient-sensing pathways to regulate important aspects of cell growth.