The ACC synthase TOE sequence is required for interaction with ETO1 family proteins and destabilization of target proteins

The Arabidopsis ETO1 protein is a negative regulator of ethylene biosynthesis. It specifically inhibits the enzyme activity of type 2 1-aminocyclopropane-1-carboxylate synthases (ACC synthases or ACS) and promotes their degradation by a proteasome-dependent pathway. To further understand the function of the ETO1 family in the plant kingdom, we cloned a cDNA of LeEOL1 (Lycopersicon esculentum ETO 1- LIKE 1), an ETO1 homolog from tomato. LeEOL1 encodes a putative protein with domain architecture conserved in the Arabidopsis ETO1/EOL1/EOL2 proteins and in the predicted rice EOL proteins. LeEOL1 is expressed in leaf, stem, root, flower, and the full ripe stage of fruit, suggesting diverse regulatory roles in the development of tomato. Yeast two-hybrid analysis revealed specific interactions between LeEOL1 and type 2 ACC synthases. When the C-terminal 14 amino acids (TOE; target of ETO1) of LE-ACS3 specific to type 2 ACC synthases were fused to a type 1 ACS, LE-ACS2, at the corresponding position, it allowed LE-ACS2 to strongly interact with LeEOL1. A GFP-TOE^LE-ACS3 fusion protein expressed in rice calli and in the roots of wild-type Arabidopsis showed reduced stability compared to native GFP. However, the fluorescence of GFP-TOE^LE-ACS3 was comparable to that of the native GFP in Arabidopsis eto1-4 mutant. Furthermore, MG132 treatment significantly enhanced the fluorescence of GFP-TOE^LE-ACS3 in the roots of wild-type Arabidopsis. These results suggest that the ETO1-family-mediated ACS protein degradation pathway is conserved in both monocots and dicots, and that TOE acts as a protein destabilization signal recognized by the ETO1 protein family.* The nucleotide sequence reported will appear in the GenBank Nucleotide Sequence Database under the accession number DQ223268.The nucleotide sequence reported will appear in the GenBank Nucleotide Sequence Database under the accession number DQ223268     

The leukemia associated <Emphasis Type="Italic">ETO</Emphasis> nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

Background  

The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.     

Altered HepG2 cell models using etomoxir versus <Emphasis Type="Italic">tert</Emphasis>-butylhydroperoxide

Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37°C (p = 0.02, p < 0.001 and p < 0.001, respectively), but to a larger extent in the presence of ETO or TBOOH. The alteration was generally highly dependent on the ETO concentration, time, and temperature. At 37°C 24 h after (T24h), regarding ETO concentration, R2 correlation ratio was 0.65 (p < 0.001), 0.70 (p < 0.001), and 0.89 (p < 0.001) for ATP levels, protein content, and viability, respectively. The lowest ETO concentration producing a significant effect was 0.25 mM. Concerning time dependency (i.e., T24h versus after 5 h (T5h)), at 37°C with ETO, ATP level continued to significantly decrease between T5h and T24h. In a similar way, at 37°C, the MTT viability test decrease was accelerated only between T5h and T24h for ETO concentrations higher than 0.5 mM (p = 0.016 and p = 0.0001 for 0.75 and 1 mM, respectively). On the contrary, with TBOOH, comparing T24h versus T5h, cellular indicators were improved but generally remained lower than MEM without any interfering agent at T24h, suggesting that TBOOH action was time limited probably in relation with its oxidation in cell medium. This study confirms the interest of altered ETO cell model to screen agents (or formulation) prone to prevent or treat energetic depletion in relation with oxidative stress.     

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 ETO1^F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1^F466I 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.     

Brassinosteroid induction of AtACS4 encoding an auxin-responsive 1-aminocyclopropane-1-carboxylate synthase 4 in Arabidopsis seedlings

Brassinosteroid (BR), an endogenous steroid growth regulator of higher plants, enhances expansion and division of the cell in a number of plant species. It has been recently reported that a shared auxin–BR signalling pathway is involved in the seedling growth in Arabidopsis . Here, we show that BR specifically enhanced the expression of AtACS4 , which encodes an auxin-responsive ACC synthase 4, by a distinct temporal induction mechanism compared with that of IAA in etiolated Arabidopsis seedlings. This BR induction of AtACS4 was undetectable in the light-grown seedlings. In addition, BR failed to activate the AtACS4 gene in auxin-resistant1 ( axr1-3 ) and auxin-resistant2 ( axr2-1 ), both of which are auxin-resistant mutants. Thus, it appears that there is a possible regulatory link between light, auxin and BR to control ethylene synthesis in Arabidopsis young seedlings. Analysis of transgenic Arabidopsis plants harbouring AtACS4::GUS fusion revealed the AtACS4 promoter-driven GUS activity in the highly elongating zone of the hypocotyls in response to BR treatment. Furthermore, Arabidopsis plants homozygous for the T-DNA insertion in the AtACS4 gene exhibited longer hypocotyls and roots than those of control seedlings. Taken together, these results suggest that the BR-induced ethylene production may participate in the elongation growth response in early seedling development of Arabidopsis .     

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase,isolated from <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Background  

Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.     

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors <Emphasis Type="Italic">ETR1</Emphasis> and <Emphasis Type="Italic">ERS1</Emphasis>

Background  

The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations.     

Co-suppression of the <Emphasis Type="Italic">EIN2</Emphasis>-homology gene <Emphasis Type="Italic">LeEIN2</Emphasis> inhibits fruit ripening and reduces ethylene sensitivity in tomato

EIN2 (ethylene insensitive 2) is a very important component in the ethylene signal transduction pathway. Recently, the genomic DNA and full-length cDNA of LeEIN2 (tomato EIN2) have been isolated in our laboratory. To reveal the function of LeEIN2, transgenic tomato plants with reduced expression levels of LeEIN2 were produced. The fruit ripening and expressions of ripening-related genes encoding polygalacturonase and TomLoxB were inhibited in the LeEIN2-silenced transgenic plants compared to the wild-type Ailsa Craig. In the seedling ethylene response assay, the transgenic tomato plants with reduced LeEIN2 expression exhibited ethylene insensitivity. These results indicate that LeEIN2 plays a critical role in regulating tomato fruit ripening and is a positive regulator in ethylene signal transduction pathway.     

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentallyand environmentally. To investigate the regulation of ACC synthasegene expression, the promoters of Arabidopsis ACS genes, AtACS4,AtACS5, and AtACS7, were fused to a GUS reporter gene, and therecombinant transgenes were introduced into Arabidopsis to producethree groups of AtACS::GUS transgenic plants. Histochemic andfluorometric study of these transgenic plants revealed thatpromoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinatedseedlings. AtACS5 has the highest promoter activity in leavesof 2-week-old light-grown seedlings among the three AtACS genesstudied. In the mature leaves, AtACS4 and AtACS7 genes are expressedin both veins and areoles, whereas AtACS5 is expressed at ahigher level in the areoles and epidermal cells surroundingtrichomes. The promoter activities of all these AtACS genesare found in the reproductive organs. AtACS5 and AtACS7 arehighly expressed in petals, sepals, carpels, stamens, caulineleaves, inflorescence stems, and siliques, while AtACS4 expressionis undetectable in the petals of open flowers. All three AtACSgenes are expressed in root tissue. In the 2-week-old light-grownArabidopsis, the AtACS4 promoter is responsive to the planthormones IAA, ethylene, and ABA, and to darkness and wounding;the AtACS5 promoter to IAA, ABA, salt, high temperature, andwounding; and the AtACS7 promoter to GA₃, ethylene, and ABA,and to darkness and salt. Low-temperature treatment abolishesthe darkness-induced AtACS7 gene expression, but not that ofAtACS4. Each AtACS gene has a unique expression profile duringgrowth and development. It appears that at any developmentalstage or any growth period of Arabidopsis, there is always amember of AtACS multigene family that is actively expressed. Key words: ACC synthase, Arabidopsis, ethylene, gene expression, GUS histochemical staining, reporter, stress treatments     

Information flow during gene activation by signaling molecules: ethylene transduction in Arabidopsis cells as a study system

Background  

We study root cells from the model plant Arabidopsis thaliana and the communication channel conformed by the ethylene signal transduction pathway. A basic equation taken from our previous work relates the probability of expression of the gene ERF1 to the concentration of ethylene.     

Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor <Emphasis Type="Italic">TERF2</Emphasis>/<Emphasis Type="Italic">LeERF2</Emphasis> is modulated by ethylene biosynthesis

Increasing numbers of investigations indicate that ethylene response factor (ERF) proteins play important roles in plant stress responses via interacting with GCC box and/dehydration-responsive element/C-repeat to modulate expression of downstream genes, but the detailed regulatory mechanism is not well elucidated. Revealing the modulation pathway of ERF proteins in response to stresses is vital. Previously, we showed that tomato ERF protein TERF2/LeERF2 is ethylene inducible, and ethylene production is suppressed in antisense TERF2/LeERF2 tomatoes, suggesting that TERF2/LeERF2 functions as a positive regulator in ethylene biosynthesis. In this paper, we report that regulation of TERF2/LeERF2 in ethylene biosynthesis is associated with enhanced freezing tolerance of tobacco and tomato. Analysis of gene expression showed that cold slowly induces expression of TERF2/LeERF2 in tomato, implying that TERF2/LeERF2 may be involved in cold response through ethylene modulation. To test the hypothesis, we first observed that overexpressing TERF2/LeERF2 tobaccos not only enhances freezing tolerance via activating expression of cold-related genes, but also significantly reduces electrolyte leakage. In addition, with treatment of ethylene biosynthesis inhibitor or ethylene receptor antagonist, we then showed that blockage of ethylene biosynthesis or the ethylene signaling pathway decreases freezing tolerance of overexpressing TERF2/LeERF2 tobaccos. Moreover, the results from tomatoes showed that overexpressing TERF2/LeERF2 tomatoes enhances while antisense TERF2/LeERF2 transgenic lines decreases freezing tolerance, and application of ethylene precursor 1-aminocyclopropane-1-carboxylic acid restored freezing tolerance of antisense lines. Therefore our results establish that TERF2/LeERF2 enhances freezing tolerance of plants through ethylene biosynthesis and the ethylene signaling pathway.     

Cloning and expression of <Emphasis Type="Italic">R-Spondin1</Emphasis>in different vertebrates suggests a conserved role in ovarian development

Background  

R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway.     

Regulation of genes affecting body size and innate immunity by the DBL-1/BMP-like pathway in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Bone morphogenetic proteins (BMPs) are members of the conserved transforming growth factor β (TGFβ superfamily, and play many developmental and homeostatic roles. In C. elegans, a BMP-like pathway, the DBL-1 pathway, controls body size and is involved in innate immunity. How these functions are carried out, though, and what most of the downstream targets of this pathway are, remain unknown.     

Diversity and selective sweep in the OsAMT1;1 genomic region of rice

Background  

Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives.     

Inactivation of NMD increases viability of <Emphasis Type="Italic">sup45</Emphasis> nonsense mutants in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.