Arabidopsis JMJ17 promotes cotyledon greening during de-etiolation by repressing genes involved in tetrapyrrole biosynthesis in etiolated seedlings.

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.

Dimerization of the ETO1 family proteins plays a crucial role in regulating ethylene biosynthesis in Arabidopsis thaliana.

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.

CHITINASE LIKE1 Regulates Root Development of Dark-Grown Seedlings by Modulating Ethylene Biosynthesis in Arabidopsis thaliana.

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) ethylene overproducer (eto) seedlings. Here, we describe our isolation and characterization of an Arabidopsis revert to eto1 9 (ret9) mutant, which showed reduced sensitivity to acsinones in etiolated eto1 seedlings. Map-based cloning of RET9 revealed an amino acid substitution in CHITINASE LIKE1 (CTL1), which is required for cell wall biogenesis and stress resistance in Arabidopsis. Etiolated seedlings of ctl1ret9 showed short hypocotyls and roots, which were augmented in combination with eto1-4. Consistently, ctl1ret9 seedlings showed enhanced sensitivity to exogenous ACC to suppress primary root elongation as compared with the wild type. After introducing ctl1ret9 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 ctl1ret9 seedlings. Furthermore, a mild yet significantly increased ethylene level in ctl1 mutants was related to elevated mRNA level and activity of ACC oxidase (ACO). Moreover, genes associated with ethylene biosynthesis (ACO1 and ACO2) and response (ERF1 and EDF1) were upregulated in etiolated ctl1ret9 seedlings. By characterizing a new recessive allele of CTL1, 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.

ETHYLENE RESPONSE FACTOR 96 positively regulates Arabidopsis resistance to necrotrophic pathogens by direct binding to GCC elements of jasmonate - and ethylene-responsive defence genes.

The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.

The Histone Acetyltransferase Gcn5 Regulates ncRNA-ICR1 and FLO11 Expression during Pseudohyphal Development in Saccharomyces cerevisiae.

Filamentous growth is one of the key features of pathogenic fungi during the early infectious phase. The pseudohyphal development of yeast Saccharomyces cerevisiae shares similar characteristics with hyphae elongation in pathogenic fungi. The expression of FLO11 is essential for adhesive growth and filament formation in yeast and is governed by a multilayered transcriptional network. Here we discovered a role for the histone acetyltransferase general control nonderepressible 5 (Gcn5) in regulating FLO11-mediated pseudohyphal growth. The expression patterns of FLO11 were distinct in haploid and diploid yeast under amino acid starvation induced by 3-amino-1,2,4-triazole (3AT). In diploids, FLO11 expression was substantially induced at a very early stage of pseudohyphal development and decreased quickly, but in haploids, it was gradually induced. Furthermore, the transcription factor Gcn4 was recruited to the Sfl1-Flo8 toggle sites at the FLO11 promoter under 3AT treatment. Moreover, the histone acetylase activity of Gcn5 was required for FLO11 induction. Finally, Gcn5 functioned as a negative regulator of the noncoding RNA ICR1, which is known to suppress FLO11 expression. Gcn5 plays an important role in the regulatory network of FLO11 expression via Gcn4 by downregulating ICR1 expression, which derepresses FLO11 for promoting pseudohyphal development.

Transcriptional consequence and impaired gametogenesis with high-grade aneuploidy in Arabidopsis thaliana.

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.

Investigating the phytohormone ethylene response pathway by chemical genetics.

Conventional mutant screening in forward genetics research is indispensible to understand the biological operation behind any given phenotype. However, several issues, such as functional redundancy and lethality or sterility resulting from null mutations, frequently impede the functional characterization of genetic mutants. As an alternative approach, chemical screening with natural products or synthetic small molecules that act as conditional mutagens allows for identifying bioactive compounds as bioprobes to overcome the above-mentioned issues. Ethylene is the simplest olefin and is one of the major phytohormones playing crucial roles in plant physiology. Most of the current information on how ethylene works in plants came primarily from genetic studies of ethylene mutants identified by conventional genetic screening two decades ago. However, we lack a complete picture of functional interaction among components in the ethylene pathway and cross talk of ethylene with other phytohormones. Here, we describe our methodology for using chemical genetics to identify small molecules that interfere with the ethylene response. We set up a phenotype-based screening platform and a reporter gene-based system for verification of the hit compounds identified by chemical screening. We have successfully identified small molecules affecting the ethylene phenotype in etiolated seedlings and showed that a group of structurally similar compounds are novel inhibitors of ACC synthase, a rate-limiting enzyme in the ethylene biosynthesis pathway.

Dissociation of the H3K36 demethylase Rph1 from chromatin mediates derepression of environmental stress-response genes under genotoxic stress in Saccharomyces cerevisiae.

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.

A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.

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 (cesa6(ret8);eto1-4) and ret41 (det2(ret41);eto1-5), respectively. Etiolated seedlings of both ret8 and ret41 exhibit short hypocotyls and roots. Ethylene levels were similar in etiolated cesa6(ret8) 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 cesa6(ret8) and det2(ret41). However, together with eto1, cesa6(ret8) and det2(ret41) 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.

Ethylene response pathway is essential for ARABIDOPSIS A-FIFTEEN function in floral induction and leaf senescence.

ARABIDOPSIS A-FIFTEEN (AAF) encodes a plastid protein and was originally identified as a SENESCENCE-ASSOCIATED GENE. Previously, we found that overexpression of AAF (AAF-OX) in Arabidopsis led to accumulated reactive oxygen species and promoted leaf senescence induced by oxidative stress, which was suppressed by a null mutant, ein2-5, in ethylene response pathway. Whether AAF function is involved in ethylene biosynthesis and/or the response pathway remained unknown. Here we show that neither overexpression (AAF-OX) nor a null mutant (aaf-KO) of AAF generates a higher level of ethylene than the wild type and display a typical triple-response phenotype in etiolated seedlings treated with 1-aminocyclopropane-1-carboxylic acid (ACC). Nevertheless, ein2-5 suppresses the phenotypes of early flowering and age-dependent leaf senescence in AAF-OX plants. We reveal that a functional ethylene response is essential for AAF function in leaf senescence and floral induction, but AAF is unlikely a regulatory component integral to the ethylene pathway.

Role of ARABIDOPSIS A-FIFTEEN in regulating leaf senescence involves response to reactive oxygen species and is dependent on ETHYLENE INSENSITIVE2.

Leaf senescence is a highly regulated developmental process that is coordinated by several factors. Many senescence-associated genes (SAGs) have been identified, but their roles during senescence remain unclear. A sweet potato (Ipomoea batatas) SAG, named SPA15, whose function was unknown, was identified previously. To understand the role of SPA15 in leaf senescence further, the orthologue of SPA15 in Arabidopsis thaliana was identified and characterized, and it was named ARABIDOPSIS A-FIFTEEN (AAF). AAF was expressed in early senescent leaves and in tissues with highly proliferative activities. AAF was localized to the chloroplasts by transient expression in Arabidopsis mesophyll protoplasts. Overexpression of AAF (AAF-OX) in Arabidopsis promoted, but the T-DNA insertion mutant (aaf-KO), delayed age-dependent leaf senescence. Furthermore, stress-induced leaf senescence caused by continuous darkness was enhanced in AAF-OX but suppressed in aaf-KO. Transcriptome analysis of expression profiles revealed up-regulated genes related to pathogen defence, senescence, and oxidative stress in 3-week-old AAF-OX plants. Indeed, elevated levels of reactive oxygen species (ROS) and enhanced sensitivity to oxidative and dark stress were apparent in AAF-OX but reduced in aaf-KO. ETHYLENE INSENSITIVE2 (EIN2) was required for the dark- and ROS-induced senescence phenotypes in AAF-OX and the induction of AAF expression by treatment with the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid. The results indicate the functional role of AAF is an involvement in redox homeostasis to regulate leaf senescence mediated by age and stress factors during Arabidopsis development.

The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1.

The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

Identification of novel inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase by chemical screening in Arabidopsis thaliana.

Ethylene is a gaseous hormone important for adaptation and survival in plants. To further understand the signaling and regulatory network of ethylene, we used a phenotype-based screening strategy to identify chemical compounds interfering with the ethylene response in Arabidopsis thaliana. By screening a collection of 10,000 structurally diverse small molecules, we identified compounds suppressing the constitutive triple response phenotype in the ethylene overproducer mutant eto1-4. The compounds reduced the expression of a reporter gene responsive to ethylene and the otherwise elevated level of ethylene in eto1-4. Structure and function analysis revealed that the compounds contained a quinazolinone backbone. Further studies with genetic mutants and transgenic plants involved in the ethylene pathway showed that the compounds inhibited ethylene biosynthesis at the step of converting S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Biochemical studies with in vitro activity assay and enzyme kinetics analysis indicated that a representative compound was an uncompetitive inhibitor of ACC synthase. Finally, global gene expression profiling uncovered a significant number of genes that were co-regulated by the compounds and aminoethoxyvinylglycine, a potent inhibitor of ACC synthase. The use of chemical screening is feasible in identifying small molecules modulating the ethylene response in Arabidopsis seedlings. The discovery of such chemical compounds will be useful in ethylene research and can offer potentially useful agrochemicals for quality improvement in post-harvest agriculture.