Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome  b ₅ (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.     

Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1-349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K(d), 117 nM) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K(d), 1.38 μM). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.     

Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis

Ethylene is an important regulator of plant growth, development and responses to environmental stresses. Arabidopsis perceives ethylene through five homologous receptors that negatively regulate ethylene responses. RTE1, a novel gene conserved in plants, animals and some protists, was recently identified as a positive regulator of the ETR1 ethylene receptor. Here, we genetically analyze the dependence of ETR1 on RTE1 in order to obtain further insight into RTE1 function. The function of RTE1 was found to be independent and distinct from that of RAN1, which encodes a copper transporter required for ethylene receptor function. We tested the ability of an rte1 loss-of-function mutation to suppress 11 etr1 ethylene-binding domain mis-sense mutations, all of which result in dominant ethylene insensitivity due to constitutive signaling. This suppression test uncovered two classes of etr1 mutations -RTE1-dependent and RTE1-independent. The nature of these mutations suggests that the ethylene-binding domain is a possible target of RTE1 action. Based on these findings, we propose that RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain.     

RTE1 is a Golgi-associated and ETR1-dependent negative regulator of ethylene responses

Arabidopsis (Arabidopsis thaliana) RTE1 encodes a membrane protein and negatively regulates ethylene responses. Genetic and transformation studies suggest that the function of the wild-type RTE1 is primarily dependent on ETR1 and can be independent on the other receptors. Ethylene insensitivity caused by the overexpression of RTE1 is largely masked by the etr1-7 mutation, but not by any other receptor mutations. The wild-type ETR1 N terminus is sufficient to the activation of the RTE1 function and the ectopic expression of etr1(1-349) restored ethylene insensitivity conferred by 35SgRTE1 in etr1-7. The RTE1 N terminus is not essential to the etr1-2 function and the expression of rte1(NDelta49), which has an N-terminal deletion of 49 amino acid residues, restored ethylene insensitivity in etr1-2 rte1-2. The ectopic expression of GREEN FLUORESCENT PROTEIN (GFP)-RTE1 conferred ethylene insensitivity in wild type and the GFP fusion displayed fast movement within the cytoplasm. The GFP-RTE1 and EYFP-NAG proteins colocalized and the Brefeldin A treatment caused aggregation of GFP-RTE1, suggesting RTE1 is a Golgi-associated protein. Our results suggest specificity of the RTE1 function to ETR1 and that endomembranes may play a role in the ethylene signal transduction.     

Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues

Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.     

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane‐bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor‐interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi‐fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1‐1 and etr1‐2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.     

Metabolomic approaches reveal that cell wall modifications play a major role in ethylene-mediated resistance against Botrytis cinerea

In Arabidopsis, resistance to the necrotrophic fungus Botrytis cinerea is conferred by ethylene via poorly understood mechanisms. Metabolomic approaches compared the responses of the wild‐type, the ethylene‐insensitive mutant etr1‐1, which showed increased susceptibility, and the constitutively active ethylene mutants ctr1‐1 and eto2 both exhibited decreased susceptibility to B. cinerea. Fourier transform–infrared (FT‐IR) spectroscopy demonstrated reproducible biochemical differences between treatments and genotypes. To identify discriminatory mass‐to‐charge ratios (m/z) associated with resistance, discriminant function analysis was employed on spectra derived from direct injection electrospray ionisation‐mass spectrometry on the derived principal components of these data. Ethylene‐modulated m/z were mapped onto Arabidopsis biochemical pathways and many were associated with hydroxycinnamate and monolignol biosynthesis, both linked to cell wall modification. A high‐resolution linear triple quadrupole‐Orbitrap hybrid system confirmed the identity of key metabolites in these pathways. The contribution of these pathways to defence against B. cinerea was validated through the use of multiple Arabidopsis mutants. The FT‐IR microspectroscopy indicated that spatial accumulation of hydroxycinnamates and monolignols at the cell wall to confine disease was linked ot ethylene. These data demonstrate the power of metabolomic approaches in elucidating novel biological phenomena, especially when coupled to validation steps exploiting relevant mutant genotypes.     

Possible modulation of Arabidopsis ETR1 N-terminal signaling by CTR1

The mitogen-activated protein kinase kinase kinase (MAPKKK) Constitutive Triple-Response1 (CTR1) plays a key role in mediating ethylene receptor signaling via its N-terminal interaction with the ethylene receptor C-terminal histidine kinase (HK) domain. Loss-of-function mutations of CTR1 prevent ethylene receptor signaling, and corresponding ctr1 mutants show a constitutive ethylene response phenotype. We recently reported in Plant Physiology that expression of the truncated ethylene receptor Ethylene Response1 (ETR1) isoforms etr11-349 and dominant ethylene-insensitive etr1-11-349, lacking the C-terminal HK and receiver domains, both suppressed the ctr1 mutant phenotype. Therefore, the ETR1 N terminus is capable of receptor signaling independent of CTR1. The constitutive ethylene response phenotype is stronger for ctr1-1 than ctr1-1 lines expressing the etr11-349 transgene, so N-terminal signaling by the full-length but not truncated ETR1 is inhibited by ctr1-1. We address possible modulations of ETR1 N-terminal signaling with docking of CTR1 on the ETR1 HK domain.     

Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii

In many eukaryotes, endoplasmic reticulum (ER ) stress activates the unfolded protein response (UPR ) via the transmembrane endoribonuclease IRE 1 to maintain ER homeostasis. The ER stress response in microalgae has not been studied in detail. Here, we identified Chlamydomonas reinhardtii IRE 1 (CrIRE 1 ) and characterized two independent knock‐down alleles of this gene. CrIRE 1 is similar to IRE 1s identified in budding yeast, plants, and humans, in terms of conserved domains, but differs in having the tandem zinc‐finger domain at the C terminus. CrIRE 1 was highly induced under ER stress conditions, and the expression of a chimeric protein consisting of the luminal N‐terminal region of CrIRE 1 fused to the cytosolic C‐terminal region of yeast Ire1p rescued the yeast ?ire1 mutant. Both allelic ire1 knock‐down mutants ire1‐1 and ire1‐2 were much more sensitive than their parental strain CC ‐4533 to the ER stress inducers tunicamycin, dithiothreitol and brefeldin A. Treatment with a low concentration of tunicamycin resulted in growth arrest and cytolysis in ire1 mutants, but not in CC ‐4533 cells. Furthermore, in the mutants, ER stress marker gene expression was reduced, and reactive oxygen species (ROS ) marker gene expression was increased. The survival of ire1 mutants treated with tunicamycin improved in the presence of the ROS scavenger glutathione, suggesting that ire1 mutants failed to maintain ROS levels under ER stress. Together, these results indicate that CrIRE 1 functions as an important component of the ER stress response in Chlamydomonas, and suggest that the ER stress sensor IRE 1 is highly conserved during the evolutionary history.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed.     

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence

Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.