Summary
Arabidopsis histone H3 lysine 4 (H3K4) demethylases play crucial roles in several developmental processes, but their involvement in seedling establishment remain unexplored.
Here, we show ...that Arabidopsis JUMONJI DOMAIN‐CONTAINING PROTEIN17 (JMJ17), an H3K4me3 demethylase, is involved in cotyledon greening during seedling establishment. Dark‐grown seedlings of jmj17 accumulated a high concentration of protochlorophyllide, an intermediate metabolite in the tetrapyrrole biosynthesis (TPB) pathway that generates chlorophyll (Chl) during photomorphogenesis. Upon light irradiation, jmj17 mutants displayed decreased cotyledon greening and reduced Chl level compared with the wild‐type; overexpression of JMJ17 completely rescued the jmj17‐5 phenotype.
Transcriptomics analysis uncovered that several genes encoding key enzymes involved in TPB were upregulated in etiolated jmj17 seedlings. Consistently, chromatin immunoprecipitation‐quantitative PCR revealed elevated H3K4me3 level at the promoters of target genes. Chromatin association of JMJ17 was diminished upon light exposure. Furthermore, JMJ17 interacted with PHYTOCHROME INTERACTING FACTOR1 in the yeast two‐hybrid assay.
JMJ17 binds directly to gene promoters to demethylate H3K4me3 to suppress PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C expression and TPB in the dark. Light results in de‐repression of gene expression to modulate seedling greening during de‐etiolation. Our study reveals a new role for histone demethylase JMJ17 in controlling cotyledon greening in etiolated seedlings during the dark‐to‐light transition.
See also the Commentary on this article by Woodson, 231: 907–909.
Summary
ETHYLENE OVERPRODUCER1 (ETO1), ETO1‐LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric‐a‐brac (BTB) protein family that collectively regulate type‐2 ...1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type‐2 ACS activity via protein–protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue‐specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain‐swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1‐5 generates a substitution of phenylalanine with an isoleucine in ETO1F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1F466I in Arabidopsis results in a constitutive triple response phenotype in dark‐grown seedlings. Our findings reveal the mechanistic role of protein–protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.
Significance Statement
The ETO1 family proteins function collectively as negative regulators of ethylene biosynthesis by promoting protein degradation of type‐2 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase via differential association with CULLIN3 (CUL3)‐RING E3 ubiquitin ligases (CRL3) in Arabidopsis thaliana. This study provides a mechanistic view of such a regulation, which is dependent on the ability of ETO1 to form homodimers and dimerize with EOL1 and EOL2 to incorporate type‐2 ACC synthase into the CRL3ETO1/EOL complex for protein ubiquitination followed by degradation via the 26S proteasome.
The plant hormone ethylene plays a regulatory role in development in light- and dark-grown seedlings. We previously isolated a group of small-molecule compounds with a quinazolinone backbone, which ...were named acsinones (for ACC synthase inhibitor quinazolinones), that act as uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). Thus, the triple response phenotype, which consists of shortened hypocotyls and roots, radial swelling of hypocotyls and exaggerated curvature of apical hooks, was suppressed by acsinones in dark-grown (etiolated)
(
) seedlings. Here, we describe our isolation and characterization of an Arabidopsis
(
) mutant, which showed reduced sensitivity to acsinones in etiolated
seedlings. Map-based cloning of
revealed an amino acid substitution in
(
), which is required for cell wall biogenesis and stress resistance in Arabidopsis. Etiolated seedlings of
showed short hypocotyls and roots, which were augmented in combination with
. Consistently,
seedlings showed enhanced sensitivity to exogenous ACC to suppress primary root elongation as compared with the wild type. After introducing
to mutants completely insensitive to ethylene, genetic analysis indicated that an intact ethylene response pathway is essential for the alterations in root and apical hook but not hypocotyl in etiolated
seedlings. Furthermore, a mild yet significantly increased ethylene level in
mutants was related to elevated mRNA level and activity of ACC oxidase (ACO). Moreover, genes associated with ethylene biosynthesis (
and
) and response (
and
) were upregulated in etiolated
seedlings. By characterizing a new recessive allele of
, we reveal that CTL1 negatively regulates ACO activity and the ethylene response, which thus contributes to understanding a role for ethylene in root elongation in response to perturbed cell wall integrity.
Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B ...monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.
Aneuploidy features a numerical chromosome variant that the number of chromosomes in the nucleus of a cell is not an exact multiple of the haploid number, which may have an impact on morphology and ...gene expression. Here we report a tertiary trisomy uncovered by characterizing a T-DNA insertion mutant (aur2-1/+) in the Arabidopsis (Arabidopsis thaliana) AURORA2 locus. Whole-genome analysis with DNA tiling arrays revealed a chromosomal translocation linked to the aur2-1 allele, which collectively accounted for a tertiary trisomy 2. Morphologic, cytogenetic and genetic analyses of aur2-1 progeny showed impaired male and female gametogenesis to various degrees and a tight association of the aur2-1 allele with the tertiary trisomy that was preferentially inherited. Transcriptome analysis showed overlapping and distinct gene expression profiles between primary and tertiary trisomy 2 plants, particularly genes involved in response to stress and various types of external and internal stimuli. Additionally, transcriptome and gene ontology analyses revealed an overrepresentation of nuclear-encoded organelle-related genes functionally involved in plastids, mitochondria and peroxisomes that were differentially expressed in at least three if not all Arabidopsis trisomics. These observations support a previous hypothesis that aneuploid cells have higher energy requirement to overcome the detrimental effects of an unbalanced genome. Moreover, our findings extend the knowledge of the complex nature of the T-DNA insertion event influencing plant genomic integrity by creating high-grade trisomy. Finally, gene expression profiling results provide useful information for future research to compare primary and tertiary trisomics for the effects of aneuploidy on plant cell physiology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase ...regulates INO1 gene activation in part via Gcn5/SAGA complex‐mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA‐bound activators, and in its role in modifying histones and recruiting TATA‐binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA‐bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter‐specific requirements.
Cells respond to environmental signals by altering gene expression through transcription factors. Rph1 is a histone demethylase containing a Jumonji C (JmjC) domain and belongs to the C(2)H(2) ...zinc-finger protein family. Here we investigate the regulatory network of Rph1 in yeast by expression microarray analysis. More than 75% of Rph1-regulated genes showed increased expression in the rph1-deletion mutant, suggesting that Rph1 is mainly a transcriptional repressor. The binding motif 5'-CCCCTWA-3', which resembles the stress response element, is overrepresented in the promoters of Rph1-repressed genes. A significant proportion of Rph1-regulated genes respond to DNA damage and environmental stress. Rph1 is a labile protein, and Rad53 negatively modulates Rph1 protein level. We find that the JmjN domain is important in maintaining protein stability and the repressive effect of Rph1. Rph1 is directly associated with the promoter region of targeted genes and dissociated from chromatin before transcriptional derepression on DNA damage and oxidative stress. Of interest, the master stress-activated regulator Msn2 also regulates a subset of Rph1-repressed genes under oxidative stress. Our findings confirm the regulatory role of Rph1 as a transcriptional repressor and reveal that Rph1 might be a regulatory node connecting different signaling pathways responding to environmental stresses.
Multiple covalent modifications exist in the amino-terminal tails of core histones, but whether a relationship exists between them is unknown. We examined the relationship between serine 10 ...phosphorylation and lysine 14 acetylation in histone H3 and have found that, in vitro, several HAT enzymes displayed increased activity on H3 peptides bearing phospho-Ser-10. This augmenting effect of Ser-10 phosphorylation on acetylation by yGcn5 was lost by substitution of alanine for arginine 164 Gcn5(R164A), a residue close to Ser-10 in the structure of the ternary tGcn5/CoA/histone H3 complex. Gcn5(R164A) had reduced activity in vivo at a subset of Gcn5-dependent promoters, and, strikingly, transcription of this same subset of genes was also impaired by substitution of serine 10 to alanine in the histone H3 tail. These observations suggest that transcriptional regulation occurs by multiple mechanistically linked covalent modifications of histones.
Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Δ, gis1Δ, rph1Δ, jhd1Δ, and jhd2Δ (yjr119cΔ)) ...of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Δ. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Δ and jhd2Δ strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Δ and jhd1Δ strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.
•Use of chemical genetics to explore the roles of ethylene in seedling development.•Identification of ret mutants involved in hypocotyl elongation in etiolated seedlings.•Mutations in cellulose ...synthase6 and de-etiolated2 uncovered in 2 ret mutants.•Cellulose synthesis and brassinolides are required in regulating hypocotyl growth.
The development of juvenile seedlings after germination is critical for the initial establishment of reproductive plants. Ethylene plays a pivotal role in the growth of seedlings under light or dark during early development. Previously, we identified small molecules sharing a quinazolinone backbone that suppressed the constitutive triple response phenotype in dark-grown eto1-4 seedlings. We designated these small molecules as ACSinhibitor quinazolinones (acsinones), which were uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase. To explore the additional roles of acsinones in plants, we screened and identified 19 Arabidopsis mutants with reduced sensitivity to acsinone7303, which were collectively named revert to eto1 (ret) because of their recovery of the eto1 phenotype. A map-based cloning approach revealed that CELLULOSE SYNTHASE6 (CESA6) and DE-ETIOLATED2 (DET2) were mutated in ret8 (cesa6ret8;eto1-4) and ret41 (det2ret41;eto1-5), respectively. Etiolated seedlings of both ret8 and ret41 exhibit short hypocotyls and roots. Ethylene levels were similar in etiolated cesa6ret8 and det2-1 and in eto1 mutants treated with acsinone7303. Chemical inhibitors of ethylene biosynthesis and perception did not significantly suppress the etiolated phenotype of cesa6ret8 and det2ret41. However, together with eto1, cesa6ret8 and det2ret41 exhibited an enhanced phenotype in the hypocotyls and apical hooks of etiolated seedlings. These results confirm that, in addition to ethylene, cellulose synthesis and brassinolides can independently contribute to modulate hypocotyl development in young seedlings.
PDF
