Mechanisms,origin and heredity of Glu-1Ay silencing in wheat evolution and domestication

Key message

Allotetraploidization drives Glu-1Ay silencing in polyploid wheat.

Abstract

The high-molecular-weight glutenin subunit gene, Glu-1Ay, is always silenced in common wheat via elusive mechanisms. To investigate its silencing and heredity during wheat polyploidization and domestication, the Glu-1Ay gene was characterized in 1246 accessions containing diploid and polyploid wheat worldwide. Eight expressed Glu-1Ay alleles (in 71.81% accessions) and five silenced alleles with a premature termination codon (PTC) were identified in Triticum urartu; 4 expressed alleles (in 41.21% accessions), 13 alleles with PTCs and 1 allele with a WIS 2-1A retrotransposon were present in wild tetraploid wheat; and only silenced alleles with PTC or WIS 2-1A were in cultivated tetra- and hexaploid wheat. Both the PTC number and position in T. urartu Glu-1Ay alleles (one in the N-terminal region) differed from its progeny wild tetraploid wheat (1–5 PTCs mainly in the repetitive domain). The WIS 2-1A insertion occurred?~?0.13 million years ago in wild tetraploid wheat, much later than the allotetraploidization event. The Glu-1Ay alleles with PTCs or WIS 2-1A that arose in wild tetraploid wheat were fully succeeded to cultivated tetraploid and hexaploid wheat. In addition, the Glu-1Ay gene in wild einkorn inherited to cultivated einkorn. Our data demonstrated that the silencing of Glu-1Ay in tetraploid and hexaploid wheat was attributed to the new PTCs and WIS 2-1A insertion in wild tetraploid wheat, and most silenced alleles were delivered to the cultivated tetraploid and hexaploid wheat, providing a clear evolutionary history of the Glu-1Ay gene in the wheat polyploidization and domestication processes.

     

Characterization of novel D-hordeins from <Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>

Three genes encoding novel D-hordeins, Ns 1.3, Ns 2.6, and Ns 2.9 were isolated from Psathyrostachys juncea. The Ns 1.3 differed from Ns 2.6 and Ns 2.9 by having a shorter open reading frame (< 1.5 kb versus > 2.5 kb), and was probably not expressed as a normal protein, while the activities for Ns 2.6 and Ns 2.9 were verified by bacterial expression. Though highly similar primary structure to wheat high molecular mass glutenin subunits (HMM-GSs) and barley D-hordeins, Ns 2.6 and Ns 2.9 had more cysteine residues (nine in total) and a larger molecular mass than HMMGSs, and a longer N-terminal length than D-hordeins. Phylogenetic analysis revealed that the Ps. juncea D-hordeins were divided into Ns 1.3 type and Ns 2.6/Ns 2.9 type. Divergence times indicated that Ns 1.3 diverged the earliest from the orthologous Triticeae locus, while Ns 2.6 and Ns 2.9 and the D-hordeins from two Hordeum species diverged nearly at the same time from those loci, and the divergence between the D-hordeins of H. chilense and Ns 2.6/Ns 2.9 was more recent than between the two Hordeum species. The novel Ps. juncea D-hordeins have the potential to be very important for improving the end-use quality of wheat flours because of the presence of extra cysteine residues and longer repetitive domain, in addition they can contribute to the understanding of the evolution of Triticeae prolamins.     

Analysis of novel high-molecular-weight prolamins from <Emphasis Type="Italic">Leymus multicaulis</Emphasis> (Kar. et Kir.) Tzvelev and <Emphasis Type="Italic">L. chinensis</Emphasis> (Trin. ex Bunge) Tzvelev

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus.     

Analysis of an N-terminal deletion in subunit <Emphasis Type="Italic">a</Emphasis> of the <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP synthase

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6–20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.     

Low molecular weight glutenin subunit gene composition at <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">D3</Emphasis> loci of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and common wheat and a further view of wheat evolution

Key message

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat.

Abstract

Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01–AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

     

Overexpression of the Bx7 high molecular weight glutenin subunit on the <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> locus in a Korean wheat landrace

The Glu-B1al (Bx7^OE + By8) allele is important for bread-making quality. The allele was found in a Korean wheat landrace using specific DNA markers. Molecular analyses were conducted to identify the overexpressed Bx7 (Bx7^OE) subunit of the allele. The Korean wheat landrace (accession ID: IT166460) showed a similar protein expression level of Bx7 subunit, i.e., overexpression of Bx7 subunit towards cv. Glenlea, Canadian Western Red Spring wheat, which harbors Bx7^OE subunit of Glu-B1al as detected on SDS–PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis). In addition, 2-DE (two-dimensional electrophoresis) analysis revealed similar protein expression patterns of the Bx7 subunit regions of IT166460 and Glenlea. The proportion of Bx7 to total HMW-GSs (high molecular weight glutenin subunits) in IT166460 (56.17 ± 0.22%) was higher than that of Chinese Spring (34.75 ± 1.03%) and even that of Glenlea (46.25 ± 1.76%) as assessed by RP-HPLC (reverse-phase high-performance liquid chromatography). Overexpression of Bx7 subunit was caused by gene duplication and indels of the promoter region of the Bx7 gene. IT166460 attained the 43 bp indel of the promoter region, as did Glenlea, i.e., the amplicon size of IT166460 was the same as that of Glenlea. In addition, the nucleotides present in the duplicated gene in IT166460 were the same as those in Glenlea. Bx7^OE subunit is critical for dough strength. However, most wheat accessions harboring the subunit are distributed in America. Furthermore, most Korean wheats have little genetic variation in glutenin composition and are associated with inferior bread quality. Hence, IT166460 could be used to improve bread-making quality in the Korean wheat breeding program.     

The detection of a de novo allele of the Glu-1Dx gene in wheat–rye hybrid offspring

Key message

This study provides a link between a de novo gene and novel phenotype in wheat–rye hybrids that can be used as a model for induced de novo genetic variation.

Abstract

Wide hybridization can produce de novo DNA variation that may cause novel phenotypes. However, there is still a lack of specific links between changed genes and novel phenotypes in wide hybrids. The well-studied high-molecular-weight glutenin subunit (HMW-GS) genes in tribe Triticeae provide a useful model for addressing this issue. In this study, we investigated the feasibility of a wheat–rye hybridization method for inducing de novo phenotypes using the Glu-1Dx2.2 subunit as an example. We developed three hexaploid wheat lines with normal fertility and a Glu-1Dx2.2 variant, named Glu-1Dx2.2 ^v , derived from three F₁ hybrids. The wild-type Glu-1Dx2.2 has two direct repeats of 295 bp length separated by an intervening 101 bp in its central repetitive region. In the mutant Glu-1Dx2.2 ^v , one copy of the repeats and the intervening sequence were deleted, probably through homology-dependent illegitimate recombination (IR). This study provides a direct link between a de novo allele and novel phenotype. Our results indicate that the wheat–rye method may be a useful tool to induce de novo genetic variations that broaden the genetic diversity for wheat improvement.     

Hydrophobins from <Emphasis Type="Italic">Aspergillus</Emphasis> species cannot be clearly divided into two classes

Background

Hydrophobins are a family of small secreted proteins with a characteristic pattern of eight cysteine residues found exclusively in filamentous fungi. They have originally been divided into two classes based on their physical properties and hydropathy patterns, and are involved in the attachment of hyphae to hydrophobic structures, the formation of aerial structures and appear to be involved in pathogenicity.

Findings

Analysis of nine genome sequences from seven Aspergilli revealed fifty hydrophobins, where each species displayed between two to eight hydrophobins. Twenty of the identified hydrophobins have not previously been described from these species. Apart from the cysteines, very little amino acid sequence homology was observed. Twenty-three of the identified hydrophobins could be classified as class I hydrophobins based on their conserved cysteine spacing pattern and hydropathy pattern. However twenty-six of the identified hydrophobins were intermediate forms. Notably, a single hydrophobin, ATEG_04730, from Aspergillus terreus displayed class II cysteine spacing and had a class II hydropathy pattern.

Conclusion

Fifty hydrophobins were identified in Aspergillus, all containing the characteristic eight cysteine pattern. Aspergillus terreus exhibited both class I and class II hydrophobins. This is the first report of an Aspergillus species with the potential to express both class I and class II hydrophobins. Many of the identified hydrophobins could not directly be allocated to either class I or class II.

     

\4 integrin expression on<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> metastatic variants of lewis lung carcinoma

The expression of β4, α6, and β1 integrin subunits has been investigated on somein vitro andin vivo murine metastatic variants derived from Lewis lung carcinoma (3LL). By the use of monoclonal antibodies which recognizes different epitopes of α6, β1, and β4 subunits we demonstrate that α6 and β1 subunits are expressed in all metastatic variants of 3LL irrespective of their metastatic potential, whereas β4 subunit is expressed only in highly metastasizing cells of 3LL. Northern blots of different metastatic variants probed with β1 and β4 subunits demonstrate thata) significant amounts of β1 mRNA were detected in all metastatic variants of 3LL;b) mRNA corresponding to the described entire coding sequence of β4 subunit is expressed only on highly metastasizing cells of 3LL. We conclude that β4 subunit is specifically expressed in highly metastasizig cells of 3LL while is undetectable in lower metastasizing ones.     

Crystal structure and modeling of the tetrahedral intermediate state of methylmalonate-semialdehyde dehydrogenase (MMSDH) from <Emphasis Type="Italic">Oceanimonas doudoroffii</Emphasis>

The gene product of dddC (Uniprot code G5CZI2), from the Gram-negative marine bacterium Oceanimonas doudoroffii, is a methylmalonate-semialdehyde dehydrogenase (OdoMMSDH) enzyme. MMSDH is a member of the aldehyde dehydrogenase superfamily, and it catalyzes the NADdependent decarboxylation of methylmalonate semialdehyde to propionyl-CoA. We determined the crystal structure of OdoMMSDH at 2.9 Å resolution. Among the twelve molecules in the asymmetric unit, six subunits complexed with NAD, which was carried along the protein purification steps. OdoMMSDH exists as a stable homodimer in solution; each subunit consists of three distinct domains: an NAD-binding domain, a catalytic domain, and an oligomerization domain. Computational modeling studies of the OdoMMSDH structure revealed key residues important for substrate recognition and tetrahedral intermediate stabilization. Two basic residues (Arg103 and Arg279) and six hydrophobic residues (Phe150, Met153, Val154, Trp157, Met281, and Phe449) were found to be important for tetrahedral intermediate binding. Modeling data also suggested that the backbone amide of Cys280 and the side chain amine of Asn149 function as the oxyanion hole during the enzymatic reaction. Our results provide useful insights into the substrate recognition site residues and catalytic mechanism of OdoMMSDH.     

Antimicrobial Activities of Novel Peptides Derived from Defensin Genes of <Emphasis Type="Italic">Brassica hybrid</Emphasis> cv Pule

Plant defensins are small and basic antimicrobial peptides characterized by conserved cysteine stabilizing structure with α-helix and triple strand antiparallel β-sheet. In the present study, two novel defensin genes, designated as BhDef1 and BhDef2, was isolated from Brassica hybrid cv Pule, a native unexplored Brassicaceae species found in Thailand. The full-length cDNA of BhDef1 and BhDef2 were 240 and 258 bp encoding a 79 and 85 amino acid residues with 29 and 25 amino acid signal peptide at N-terminal, respectively. The putative BhDef1 and BhDef2 mature proteins showed significant similarity to other Brassicaceae defensins. Their secondary structure comprises of one α-helix and a triple stranded β-sheet stabilized by four disulphide bridges of eight cysteines. BhDef1 and BhDef2 also contain a highly conserved γ-core and α-core motif exhibiting antifungal activity against Colletotrichum gloeosporioides causing anthracnose disease. Six out of eight synthetic BhDef peptide derivatives showed antibacterial activity against both gram-positive bacteria and gram-negative bacteria used in this study. BhDef14, the derivative of BhDef1, showed the highest activity against two test pathogenic bacteria. This activity could probably due to a net positively charge and alpha-helical conformation which are known as the key determinant for the bacterial membrane disruption. To our knowledge, this is the first report on defensin genes isolated from B. hybrid cv Pule. The synthetic peptides designed from their sequences showed antifungal and antibacterial activity.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.     

Characterization of the complex involved in regulating V-ATPase activity of the vacuolar and endosomal membrane

Regulator of the H⁺-ATPase of the vacuolar and endosomal membranes (RAVE) is essential for the reversible assembly of H⁺-ATPase. RAVE primarily consists of three subunits: Rav1p, Rav2p and Skp1p. To characterize these subunits, in this study, four strains derived from Saccharomyces cerevisiae BY4742 were constructed with a FLAG tag on the Rav1p and Rav2p subunits. Then, the corresponding RAVE containing complex was isolated by affinity purification. Western blot and MALDI-TOF mass spectrometry analyses showed that the RAVE complex contains not only the known V₁-ATPase subunits (Vma1p and Vma2p) but also a newly found Leu1p that interacts with the RAVE subunit. Furthermore, we constructed rav1?/rav2?/vma2?/leu1-deficient recombinants by fusion PCR and homologous recombination and demonstrated that leu1 is indispensable in adjusting the microbial cell to adverse environments and that the function is similar to that of rav1/rav2 but significantly differs from that of vma2. Leu1p probably plays an important role in RAVE regulation of V-ATPase activity in conjunction with RAVE.     

Clavicipitaceous entomopathogens: new species in <Emphasis Type="Italic">Metarhizium</Emphasis> and a new genus <Emphasis Type="Italic">Nigelia</Emphasis>

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.