SummaryThe accumulation of hydrogen peroxide (H 2 O 2 ) in plants is typically associated with biotic or abiotic stresses. However, H 2 O 2 is continuously produced in cells during normal metabolism. Yet, little is known about how H 2 O 2 accumulation will affect plant metabolism in the absence of pathogens or abiotic stress. Here, we report that a de®ciency in the H 2 O 2 -scavenging enzyme, cytosolic ascorbate peroxidase (APX1), results in the accumulation of H 2 O 2 in Arabidopsis plants grown under optimal conditions. Knockout-Apx1 plants were characterized by suppressed growth and development, altered stomatal responses, and augmented induction of heat shock proteins during light stress. The inactivation of Apx1 resulted in the induction of several transcripts encoding signal transduction proteins. These were not previously linked to H 2 O 2 signaling during stress and may belong to a signal transduction pathway speci®cally involved in H 2 O 2 sensing during normal metabolism. Surprisingly, the expression of transcripts encoding H 2 O 2 scavenging enzymes, such as catalase or glutathione peroxidase, was not elevated in knockout-Apx1 plants. The expression of catalase, two typical plant peroxidases, and several different heat shock proteins was however elevated in knockout-Apx1 plants during light stress. Our results demonstrate that in planta accumulation of H 2 O 2 can suppress plant growth and development, interfere with different physiological processes, and enhance the response of plants to abiotic stress conditions. Our ®ndings also suggest that at least part of the induction of heat shock proteins during light stress in Arabidopsis is mediated by H 2 O 2 that is scavenged by APX1.

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20⌬ puf3⌬ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20⌬ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.

The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes, Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (+1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.

SummaryDormancy is an important developmental program allowing plants to withstand extended periods of extreme environmental conditions, such as low temperature or drought. Seed dormancy, bud dormancy and desiccation tolerance have been extensively studied, but little is known about the mechanisms involved in the dormancy of drought-tolerant plants, key to the survival of many plant species in arid and semi-arid environments. Subtractive PCR cloning of cDNAs from Retama raetam, a C 3 droughttolerant legume, revealed that dormancy in this plant is accompanied by the accumulation of transcripts encoding a pathogenesis-related, PR-10-like protein; a low temperature-inducible dehydrin; and a WRKY transcription factor. In contrast, non-dormant plants subjected to stress conditions contained transcripts encoding a cytosolic small heat-shock protein, HSP18; an ethylene-response transcriptional co-activator; and an early light-inducible protein. Physiological and biochemical analysis of Rubisco activity and protein in dormant and non-dormant tissues suggested a novel post-translational mechanism of regulation that may be controlled by the redox status of cells. Ultrastructural analysis of dormant plants revealed that air spaces of photosynthetic tissues contained an extracellular matrix that may function to prevent water loss. The cytosol of dormant cells appeared to be in a glassy state, limiting metabolic activity. A combination of biochemical, molecular and structural mechanisms, in association with metabolic suppression, may be key to the extreme drought tolerance of R. raetam and its acclimation to the desert ecosystem. These may enable plants to withstand long periods of drought, as well as rapidly to exit dormancy upon rainfall.

Genome-wide studies of steady-state mRNA levels revealed common principles underlying transcriptional changes in response to external stimuli. To uncover principles that govern other stages of the gene-expression response, we analyzed the translational response and its coordination with transcriptome changes following exposure to severe stress. Yeast cells were grown for 1 h in medium containing 1 M NaCl, which elicits a maximal but transient translation inhibition, and nonpolysomal or polysomal mRNA pools were subjected to DNA-microarray analyses. We observed a strong repression in polysomal association for most mRNAs, with no simple correlation with the changes in transcript levels. This led to an apparent accumulation of many mRNAs as a nontranslating pool, presumably waiting for recovery from the stress. However, some mRNAs demonstrated a correlated change in their polysomal association and their transcript levels (i.e., potentiation). This group was enriched with targets of the transcription factors Msn2/Msn4, and the translational induction of several tested mRNAs was diminished in an Msn2/Msn4 deletion strain. Genome-wide analysis of a strain lacking the high salinity response kinase Hog1p revealed that the group of translationally affected genes is significantly enriched with motifs that were shown to be associated with the AREbinding protein Pub1. Since a relatively small number of genes was affected by Hog1p deletion, additional signaling pathways are likely to be involved in coordinating the translational response to severe salinity stress.

Since the discovery that many transcriptional enhancers are transcribed into long noncoding RNAs termed "enhancer RNAs" (eRNAs), their putative role in enhancer function has been debated. Very recent evidence has indicted that some eRNAs play a role in initiating or activating transcription, possibly by helping recruit and/or stabilize binding of the general transcription machinery to the proximal promoter of their target genes. The distal enhancer of the gonadotropin hormone α-subunit gene, chorionic gonadotropin alpha (Cga), is responsible for Cga cell-specific expression in gonadotropes and thyrotropes, and we show here that it encodes two bidirectional nonpolyadenylated RNAs whose levels are increased somewhat by exposure to gonadotropin-releasing hormone but are not necessarily linked to Cga transcriptional activity. Knockdown of the more distal eRNA led to a drop in Cga mRNA levels, initially without effect on the forward eRNA levels. With time, however, the repression on the Cga increased, and the forward eRNA levels were suppressed also. We demonstrate that the interaction of the enhancer with the promoter is lost after eRNA knockdown. Dramatic changes also were seen in the chromatin, with an increase in total histone H3 occupancy throughout this region and a virtual loss of histone H3 Lys 4 trimethylation at the promoter following the eRNA knockdown. Moreover, histone H3 Lys 27 (H3K27) acetylation, which was found at both enhancer and promoter in wild-type cells, appeared to have been replaced by H3K27 trimethylation at the enhancer. Thus, the Cga eRNA mediates the physical interaction between these genomic regions and determines the chromatin structure of the proximal promoter to allow gene expression.

In eukaryotes, the switch between alternative developmental pathways is mainly attributed to a switch in transcriptional programs. A major mode in this switch is the transition between histone deacetylation and acetylation. In budding yeast, early meiosis-specific genes (EMGs) are repressed in the mitotic cell cycle by active deacetylation of their histones. Transcriptional activation of these genes in response to the meiotic signals (i.e., glucose and nitrogen depletion) requires histone acetylation. Here we follow how this regulated switch is accomplished, demonstrating the existence of two parallel mechanisms. ( Repression and regulated expression of genes by histone deacetylases (HDACs) and histone acetyltransferases (HATs) play pivotal roles in the well-being of all eukaryotic organisms. These processes are involved in the regulation of transcription, cell cycle progression, differentiation, and the development of specific tumors, as well as in controlling the life span of organisms (8, 10). Repression of specific genes is mediated by the recruitment of the HDAC complex to promoters following association with specific DNA-binding proteins (28). In addition, recruitment of HDACs to many promoters via a nontargeted, unknown mechanism leads to global deacetylation (44). A vast amount of information is emerging on repression and activation by the HDAC and HAT enzymes, but little is known about the mechanism(s) regulating the switch between deacetylation and acetylation.

Discovery of the ten-eleven translocation 1 (TET) methylcytosine dioxygenase family of enzymes, nearly 10 years ago, heralded a major breakthrough in understanding the epigenetic modifications of DNA. Initially described as catalyzing the oxidation of methyl cytosine (5mC) to hydroxymethyl cytosine (5hmC), it is now clear that these enzymes can also catalyze additional reactions leading to active DNA demethylation. The association of TET enzymes, as well as the 5hmC, with active regulatory regions of the genome has been studied extensively in embryonic stem cells, although these enzymes are expressed widely also in differentiated tissues. However, TET1 and TET3 are found as various isoforms, as a result of utilizing alternative regulatory regions in distinct tissues. Some of these isoforms, like TET2, lack the CXXC domain which probably has major implications on their recruitment to specific loci in the genome, while in certain contexts TET1 is seen paradoxically to repress transcription. In this review we bring together these novel aspects of the differential regulation of these Tet isoforms and the likely consequences on their activity.