Amino Acid Sequence Homology Research Articles

Genome-Wide Detection of SNP Markers Associated with Four Physiological Traits in Groundnut (Arachis hypogaea L.) Mini Core Collection

In order to integrate genomics in breeding and development of drought-tolerant groundnut genotypes, identification of genomic regions/genetic markers for drought surrogate traits is essential. We used 3249 diversity array technology sequencing (DArTSeq) markers for a genetic analysis of 125 ICRISAT groundnut mini core collection evaluated in 2015 and 2017 for genome-wide marker-trait association for some physiological traits and to determine the magnitude of linkage disequilibrium (LD). Marker-trait association (MTA) analysis, probability values, and percent variation modelled by the markers were calculated using the GAPIT package via the KDCompute interface. The LD analysis showed that about 36% of loci pairs were in significant LD (p < 0.05 and r2 > 0.2) and 3.14% of the pairs were in complete LD. The MTAs studies revealed 20 significant MTAs (p < 0.001) with 11 markers. Four MTAs were identified for leaf area index, 13 for canopy temperature, one for chlorophyll content and two for normalized difference vegetation index. The markers explained 20.8% to 6.6% of the phenotypic variation observed. Most of the MTAs identified on the A subgenome were also identified on the respective homeologous chromosome on the B subgenome. This could be due to a common ancestor of the A and B genome which explains the linkage detected between markers lying on different chromosomes. The markers identified in this study can serve as useful genomic resources to initiate marker-assisted selection and trait introgression of groundnut for drought tolerance after further validation.

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena.

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.

Hamster neogenin, a host-cell protein contained in a respiratory syncytial virus candidate vaccine, induces antibody responses in rabbits but not in clinical trial participants

ABSTRACT A recombinant respiratory syncytial virus (RSV) fusion glycoprotein candidate vaccine (RSV-PreF) manufactured in Chinese hamster ovary cells was developed for immunization of pregnant women, to protect newborns against RSV disease through trans-placental antibody transfer. Traces of a host-cell protein, hamster neogenin (haNEO1), were identified in purified RSV-PreF antigen material. Given the high amino-acid sequence homology between haNEO1 and human neogenin (huNEO1), there was a risk that potential vaccine-induced anti-neogenin immunity could affect huNEO1 function in mother or fetus. Anti-huNEO1 IgGs were measured by enzyme-linked immunosorbent assay in sera from rabbits and trial participants (Phase 1 and 2 trials enrolling 128 men and 500 non-pregnant women, respectively; NCT01905215/NCT02360475) collected after immunization with RSV-PreF formulations containing different antigen doses with/without aluminum-hydroxide adjuvant. In rabbits, four injections administered at 14-day intervals induced huNEO1-specific IgG responses in an antigen-dose- and adjuvant-dependent manner, which plateaued in the highest-dose groups after three injections. In humans, no vaccination-induced anti-huNEO1 IgG responses were detected upon a single immunization, as the values in vaccine and control groups fluctuated around pre-vaccination levels up to 90/360 days post-vaccination. A minority of participants had anti-huNEO1 levels ≥ assay cutoff before vaccination, which did not increase post-vaccination. Thus, despite detecting vaccine-induced huNEO1-specific responses in rabbits, we found no evidence that the candidate vaccine had induced anti-huNEO1 immunity in human adults. The antigen purification process was nevertheless optimized, and haNEO1-reduced vaccines were used in a subsequent Phase 2 trial enrolling 400 non-pregnant women (NCT02956837), in which again no vaccine-induced anti-huNEO1 responses were detected.

Simultaneous Suppression of Two Distinct Serotonin N-Acetyltransferase Isogenes by RNA Interference Leads to Severe Decreases in Melatonin and Accelerated Seed Deterioration in Rice.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthetic pathway, in which serotonin is converted into N-acetylserotonin (NAS) in plants. To date, two SNAT isogenes with low amino acid sequence homologies have been identified. Their single suppression in rice has been reported, but their double suppression in rice has not yet been attempted. Here, we generated double-suppression transgenic rice (snat1+2) using the RNA interference technique. The snat1+2 exhibited retarded seedling growths in conjunction with severe decreases in melatonin compared to wild-types and single-suppression rice plants (snat1 or snat2). The laminar angle was decreased in the snat1+2 rice compared to that of the wild-types and snat1, but was comparable to that of snat2. The reduced germination speed in the snat1+2 was comparable to that of snat2. Seed-aging testing revealed that snat1 was the most severely deteriorated, followed by snat1+2 and snat2, suggesting that melatonin is positively involved in seed longevity.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Structure, catalytic mechanism, posttranslational lysine carbamylation, and inhibition of dihydropyrimidinases.

Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-β-alanine and N-carbamyl-β-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase.

Homology between TSH-R/Tg/TPO and Hashimoto's encephalopathy autoantigens.

Hashimoto's encephalopathy (HE) is a syndrome occurring in some patients with Hashimoto's thyroiditis or, less frequently, Graves' disease. Three known autoantigens are involved in HE: alpha-enolase, dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). We searched for amino acid sequence homologies between these proteins and the three classical thyroid autoantigens (thyroperoxidase (TPO), thyroglobulin (Tg), TSH-receptor (TSH-R)), which are also expressed in the central nervous system (CNS). TSH-R shows homologies with alpha-enolase (n=4), DDAHI (n=2) and AKRIAI (n=5); of these segments, two, two and four, respectively, overlap totally or partially with epitope-containing TSH-R segments. Tg has 10 homologies with alpha-enolase, five with DDAHI, and eight with AKRIAI; epitope-containing segments of Tg overlap four, three and four segments, respectively. TPO has six segments homologous to alpha-enolase, three to DDAHI and seven to AKRIAI; of these segments, five, one and four, respectively, are located in epitope-containing parts. These data suggest that cross-reactivity between CNS autoantigens and thyroid autoantigens might contribute to the HE pathogenesis, together with other proposed mechanisms, including autoimmunity involving autoantigens common to CNS and thyroid.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1.

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

Structural insights for producing CK2α1-specific inhibitors

Casein kinase 2 catalytic subunit (CK2α) is classified into two subtypes CK2α1 and CK2α2. CK2α1 is a drug discovery target, whereas CK2α2 is an off-target of CK2α1 inhibitors. High amino acid sequence homology between these subtypes hampers efforts to produce ATP competitive inhibitors that are highly selective to CK2α1. Hematein was identified previously as a non-ATP-competitive inhibitor for CK2α1, whereas this compound acts as an ATP competitive CK2α2 inhibitor. Crystal structures of CK2α1 and CK2α2 in complex with hematein revealed distinct binding features that provide structural insights for producing CK2α1-selective inhibitors.

Allergenicity of tropomyosin of shrimp (Litopenaeus vannamei) and clam (Ruditapes philippinarum) is higher than that of fish (Larimichthys crocea) via in vitro and in vivo assessment

Tropomyosin (TM), acting as heat-stable protein, has a high homology of amino acid sequences with different sensitizations. Fish belongs to vertebrate and its TM owns less allergenicity, whereas shrimp and clam belong to invertebrate and their TM are major allergens. Moreover, the allergenicity of TM might be altered during food processing. Therefore, shrimp-TM was administered intraperitoneally to BALB/c mice to establish an allergic mouse model, to evaluate the elicitation ability of these three proteins, while a mediator-releasing RBL cell line was used to estimate the variation in the stimulation abilities. The results showed that shrimp-TM owned stronger ability in the elicitation of the anaphylactic reactivity as well as in the IgE-mediated the RBL cell line. In comparison with shrimp-TM, clam-TM showed a similar allergic reaction and T-helper 2 (Th2)-based responses from spleen cells and mediums produced by RBL cell line, while fish-TM exhibited a weaker anaphylactic reactivity based on a mouse model and produced fewer active mediums based on cell model. Furthermore, raw and boiled TM did not exhibit a significant difference in allergic reactions and the capacity of degranulation. These results indicated that shrimp-TM owned stronger stimulation ability based on allergic mouse compared with fish-TM and clam-TM, which could supply information for clinical and anaphylaxis of TM in the stages of sensitization and simulation deserve further consideration.

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.

Phenotypic and molecular characterization of Hessian fly resistance in diploid wheat, Aegilops tauschii

BackgroundThe Hessian fly (Mayetiola destructor), belonging to the gall midge family (Cecidomyiidae), is a devastating pest of wheat (Triticum aestivum) causing significant yield losses. Despite identification and characterization of numerous Hessian fly-responsive genes and associated biological pathways involved in wheat defense against this dipteran pest, their functional validation has been challenging. This is largely attributed to the large genome, polyploidy, repetitive DNA, and limited genetic resources in hexaploid wheat. The diploid progenitor Aegilops tauschii, D-genome donor of modern-day hexaploid wheat, offers an ideal surrogate eliminating the need to target all three homeologous chromosomes (A, B and D) individually, and thereby making the functional validation of candidate Hessian fly-responsive genes plausible. Furthermore, the well-annotated sequence of Ae. tauschii genome and availability of genetic resources amenable to manipulations makes the functional assays less tedious and time-consuming. However, prior to utilization of this diploid genome for downstream studies, it is imperative to characterize its physical and molecular responses to Hessian fly.ResultsIn this study we screened five Ae. tauschii accessions for their response to the Hessian fly biotypes L and vH13. Two lines were identified that exhibited a homozygous resistance response to feeding by both Hessian fly biotypes. Studies using physical measurements and neutral red staining showed that the resistant Ae. tauschii accessions resembled hexaploid wheat in their phenotypic responses to Hessian fly, that included similarities in larval developmental stages, leaf and plant growth, and cell wall permeability. Furthermore, molecular responses, characterized by gene expression profiling using quantitative real-time PCR, in select resistant Ae. tauschii lines also revealed similarities with resistant hexaploid wheat.ConclusionsPhenotypic and molecular characterization of Ae. tauschii to Hessian fly infestation revealed resistant accessions that shared similarities to hexaploid wheat. Resembling the resistant hexaploid wheat, the Ae. tauschii accessions mount an early defense strategy involving defense proteins including lectins, secondary metabolites and reactive oxygen species (ROS) radicals. Our results reveal the suitability of the diploid progenitor for use as an ideal tool for functional genomics research in deciphering the wheat-Hessian fly molecular interactions.

A Co-Expression Network in Hexaploid Wheat Reveals Mostly Balanced Expression and Lack of Significant Gene Loss of Homeologous Meiotic Genes Upon Polyploidization.

Polyploidization has played an important role in plant evolution. However, upon polyploidization, the process of meiosis must adapt to ensure the proper segregation of increased numbers of chromosomes to produce balanced gametes. It has been suggested that meiotic gene (MG) duplicates return to a single copy following whole genome duplication to stabilize the polyploid genome. Therefore, upon the polyploidization of wheat, a hexaploid species with three related (homeologous) genomes, the stabilization process may have involved rapid changes in content and expression of MGs on homeologous chromosomes (homeologs). To examine this hypothesis, sets of candidate MGs were identified in wheat using co-expression network analysis and orthology informed approaches. In total, 130 RNA-Seq samples from a range of tissues including wheat meiotic anthers were used to define co-expressed modules of genes. Three modules were significantly correlated with meiotic tissue samples but not with other tissue types. These modules were enriched for GO terms related to cell cycle, DNA replication, and chromatin modification and contained orthologs of known MGs. Overall, 74.4% of genes within these meiosis-related modules had three homeologous copies which was similar to other tissue-related modules. Amongst wheat MGs identified by orthology, rather than co-expression, the majority (93.7%) were either retained in hexaploid wheat at the same number of copies (78.4%) or increased in copy number (15.3%) compared to ancestral wheat species. Furthermore, genes within meiosis-related modules showed more balanced expression levels between homeologs than genes in non-meiosis-related modules. Taken together, our results do not support extensive gene loss nor changes in homeolog expression of MGs upon wheat polyploidization. The construction of the MG co-expression network allowed identification of hub genes and provided key targets for future studies.

Naphthoquinone Tryptophan Hybrids: A Promising Small Molecule Scaffold for Mitigating Aggregation of Amyloidogenic Proteins and Peptides.

A current challenge faced by researchers is the lack of disease-modifying therapeutics for amyloid formation that is associated with several human diseases. Although the monomeric proteins or peptides involved in various amyloidogenic diseases do not have amino acid sequence homology, there appears to be a structural correlation among the amyloid assemblies, which are responsible for distinct pathological conditions. Here, we review our work on Naphthoquinone Tryptophan (NQTrp) hybrids, a small molecule scaffold that can generically modulate neuronal and non-neuronal amyloid aggregation both in vitro and in vivo. NQTrp reduces the net amyloid load by inhibiting the process of amyloid formation and disassembling the pre-formed fibrils, both in a dose-dependent manner. As a plausible mechanism of action, NQTrp effectively forms hydrogen bonding and hydrophobic interactions, such as π-π stacking, with the vital residues responsible for the initial nucleation of protein/peptide aggregation. This review highlights the effectiveness of the NQTrp hybrid scaffold for developing novel small molecule modulators of amyloid aggregation.

Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24.

Previously, a ubiquinol-cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni-NTA resins, and IgE-binding activity was determined using IgE-western blotting and enzyme-linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE-dot blotting and IgE-ELISA with synthetic polypeptides and HDM-allergic sera. Three-dimensional (3D) structural modeling was used to analyze the position of the immuno-dominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE-ELISA and western blot analysis showed that all of the Der f-human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N-terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N-terminal reacted with IgEs from HDM-allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32-aa N-terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE-binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32-residue region of the N-terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.

DELAY OF GERMINATION1-LIKE4 acts as an inducer of seed reserve accumulation.

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION1-LIKE4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

Ion Channel Properties of a Cation Channelrhodopsin, Gt_CCR4

We previously reported a cation channelrhodopsin, Gt_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, Guillardia theta. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as Cr_ChR2 from Chlamydomonas reinhardtii, it has been proposed that a family of cryptophyte channelrhodopsin, including Gt_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known Cr_ChR2 and Gt_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by Gt_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of Cr_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of Gt_CCR4 was higher (EC50 = 0.13 mW/mm2) than that of Cr_ChR2 (EC50 = 0.80 mW/mm2) while the channel open life time (photocycle speed) was in the same range as that of Cr_ChR2 (25~30 ms for Gt_CCR4 and 10~15 ms for Cr_ChR2). This observation implies that Gt_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na+ selectivity of Gt_CCR4 in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. These results suggest that ion selectivity in Gt_CCR4 is distinct from that in Cr_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of Gt_CCR4.

Comparative Tyramide-FISH mapping of the genes controlling flavor and bulb color in Allium species revealed an altered gene order

Evolutionarily related species often share a common order of genes along homeologous chromosomes. Here we report the collinearity disruption of genes located on homeologous chromosome 4 in Allium species. Ultra-sensitive fluorescence in situ hybridization with tyramide signal amplification (tyr-FISH) allowed the visualization of the alliinase multigene family, chalcon synthase gene and EST markers on Allium cepa and Allium fistulosum chromosomes. In A. cepa, bulb alliinase, root alliinase (ALL1) and chalcon synthase (CHS-B) genes were located in the long arm but EST markers (API18 and ACM082) were located in the short arm. In A. fistulosum, all the visualized genes and markers were located in the short arm. Moreover, root alliinase genes (ALL1 and AOB249) showed contrast patterns in number of loci. We suppose that the altered order of the genes/markers is the result of a large pericentric inversion. To get insight into the evolution of the chromosome rearrangement, we mapped the bulb alliinase gene in phylogenetically close and distant species. In the taxonomic clade including A. fistulosum, A. altaicum, A. oschaninii and A. pskemense and in phylogenetically distant species A. roylei and A. nutans, the bulb alliinase gene was located on the short arm of chromosome 4 while, in A. cepa and A. schoenoprasum, the bulb alliinase gene was located on the long arm of chromosome 4. These results have encouraging implications for the further tracing of inverted regions in meiosis of interspecific hybrids and studding chromosome evolution. Also, this finding may have a practical benefit as closely related species are actively used for improving onion crop stock.

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton

BackgroundThe pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown.ResultsIn this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton.ConclusionsOur data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.