Ethylene receptor ETHYLENE RECEPTOR1 domain requirements for ethylene responses in Arabidopsis seedlings

Ethylene influences many processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptor isoforms. We used high-resolution, time-lapse imaging of dark-grown Arabidopsis seedlings to better understand the roles of each isoform in the regulation of growth in air, ethylene-stimulated nutations, and growth recovery after ethylene removal. We found that ETHYLENE RECEPTOR1 (ETR1) is both necessary and sufficient for nutations. Transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into etr1-6;etr2-3;ein4-4 triple loss-of-function mutants that have constitutive growth inhibition in air, fail to nutate in ethylene, and take longer to recover a normal growth rate when ethylene is removed. The patterns of rescue show that ETR1, ETR2, and ETHYLENE INSENSITIVE4 (EIN4) have the prominent roles in rapid growth recovery after removal of ethylene whereas ETR1 was the sole isoform that rescued nutations. ETR1 histidine kinase activity and phosphotransfer through the receiver domain are not required to rescue nutations. However, REVERSION TO SENSITIVITY1 modulates ethylene-stimulated nutations but does not modulate the rate of growth recovery after ethylene removal. Several chimeric receptor transgene constructs where domains of EIN4 were swapped into ETR1 were also introduced into the triple mutant. The pattern of phenotype rescue by the chimeric receptors used in this study supports a model where a receptor with a receiver domain is required for normal growth recovery and that nutations specifically require the full-length ETR1 receptor.     

拟南芥甲基结合蛋白基因AtMBP11启动子在种子形成和萌发中的功能鉴定

为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.     

Focus on Metabolism: Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such as vasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase (CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, which has led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerial tissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 and S. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormone biosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome of the CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues, may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functional diterpenoids in plants.Salvia miltiorrhiza, a Lamiaceae species known as red sage or tanshen, is a traditional Chinese medicinal herb that is described in Shen Nong Ben Cao Jing, the oldest classical Chinese herbal book, which dates from between 25 and 220 C.E. The lipophilic pigments from the reddish root and rhizome consist of abietane quinone diterpenoids (Nakao and Fukushima, 1934), largely tanshinone IIA, cryptotanshinone, and tanshinone I (Zhong et al., 2009). These are highly bioactive. For example, tanshinone IIA exerts vasorelaxative activity, has antiarrhythmic effects, provides protection against ischemia reperfusion injury (Zhou et al., 2005; Gao et al., 2008; Sun et al., 2008), and exhibits anticancer activities (Efferth et al., 2008; Lee et al., 2008; Wang et al., 2008; Gong et al., 2011). In addition, tanshinones have been reported to have a broad spectrum of antimicrobial activities against various plant pathogens, including rice (Oryza sativa) blast fungus Magnaporthe oryzae (Zhao et al., 2011). Although tanshinones are mainly accumulated in the roots, trace amounts of tanshinones have been detected in aerial organs as well (Hang et al., 2008).Diterpenoid biosynthesis is initiated by diterpene synthases (diTPSs), which catalyze cyclization and/or rearrangement of the general acyclic precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to form various hydrocarbon backbone structures that are precursors to more specific families of diterpenoids (Zi et al., 2014). Previous work has indicated that tanshinone biosynthesis is initiated by cyclization of GGPP to copalyl diphosphate (CPP) by a CPP synthase (SmCPS1) and subsequent further cyclization to the abietane miltiradiene by a kaurene synthase-like cyclase (SmKSL1), so named for its homology to the ent-kaurene synthases (KSs) required for GA plant hormone biosynthesis (Gao et al., 2009). Miltiradiene is a precursor to at least cryptotanshinone (Guo et al., 2013), and RNA interference (RNAi) knockdown of SmCPS1 expression reduces tanshinone production, at least in hairy root cultures (Cheng et al., 2014). The identification of SmCPS1 and SmKSL1 has been followed by that of many related diTPSs from other Lamiaceae plant species (Caniard et al., 2012; Sallaud et al., 2012; Schalk et al., 2012; Brückner et al., 2014; Pateraki et al., 2014). These largely exhibit analogous activity, particularly the CPSs, which produce CPP or the stereochemically related 8α-hydroxy-labd-13E-en-15-yl diphosphate (LDPP) rather than the enantiomeric (ent) CPP relevant to GA biosynthesis.To further investigate diterpenoid biosynthesis in S. miltiorrhiza, we report here a more thorough characterization of its diTPS family. A previously reported whole-genome shotgun sequencing survey (Ma et al., 2012) has indicated that there are at least five CPSs, although only two KSL genes in S. miltiorrhiza (Supplemental Table S1). Intriguingly, based on a combination of biochemical and genetic (RNAi gene silencing) evidence, we find that these diTPSs nevertheless account for at least four different diterpenoid biosynthetic pathways, each dependent on a unique CPS, with the KS presumably involved in GA biosynthesis seeming to be responsible for alternative diterpenoid metabolism as well. In addition, our studies clarify the evolutionary basis for the observed functional diversity, with investigation of gene structure, positive selection, molecular docking, and mutational analysis used to explore the driving force for the functional divergence of these diTPSs. Moreover, we report metabolomic analysis, also carried out with SmCPS1 RNAi lines, which enables prediction of the downstream steps in tanshinone biosynthesis.     

Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.     

WD40 Domain Divergence Is Important for Functional Differences between the Fission Yeast Tup11 and Tup12 Co-Repressor Proteins

We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains.     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

Identification of Amino Acids in the N-Terminal Domain of Corticotropin-Releasing Factor Receptor 1 that Are Important Determinants of High-Affinity Ligand Binding

Abstract : The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists : one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg⁷⁶ or Asn⁸¹ but not Gly⁸³ of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.