Molecular analysis of glycinin genes in soybean mutants for development of gene-specific markers

Soybean mutant lines that differ in 11S glycinin and 7S β-conglycinin seed storage protein subunit compositions were developed. These proteins have significant influence on tofu quality. The molecular mechanisms underlying the mutant lines are unknown. In this study, gene-specific markers for five of the glycinin genes (Gy1 to Gy5) were developed using three 11S null lines, two A₄ null Japanese cultivars, Enrei and Raiden, and a control cultivar, Harovinton. Whereas gene-specific primers produced the appropriate products in the control cultivar for the Gy1, Gy2, Gy3 and Gy5 genes, they did not amplify in mutants missing the A_1aB₂, A₂B_1a, A_1b B_1b, and A₃B₄ subunits. However, ecotype targeting induced local lesions in genomes (EcoTILLING) and sequencing analysis revealed that the absence of the A₄ peptide in the mutants is due to the same point mutation as that in Enrei and Raiden. Selection efficiency of the gene-specific primer pairs was tested using a number of breeding lines segregating for the different subunits. Primer pairs specific to each of the Gy1, Gy2, Gy3, and Gy5 genes can be used to detect the presence or absence of amplification in normal or mutant lines. The Gy4 null allele can be selected for by temperature-switch PCR (TS-PCR) for identification of the A₄ (G4) null genotypes. In comparison to protein analysis by SDS-PAGE, gene-specific markers are easier, faster and more accurate for analysis, they do not have to use seed, and can be analyzed at any plant growth stage for marker-assisted selection.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in <Emphasis Type="Italic">Flaveria linearis</Emphasis>

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO₂-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

H<Subscript>2</Subscript> inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells

H₂ is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H₂ in endothelial cells. H₂ significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-l-cysteine. H₂ inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H₂ inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H₂ probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.     

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels

Mutations in the voltage‐gated sodium channel gene SCN8A cause a broad range of human diseases, including epilepsy, intellectual disability, and ataxia. Here we describe three mouse lines on the C57BL/6J background with novel, overlapping mutations in the Scn8a DIIS4 voltage sensor: an in‐frame 9 bp deletion (Δ9), an in‐frame 3 bp insertion (?3) and a 35 bp deletion that results in a frameshift and the generation of a null allele (Δ35). Scn8a ^Δ9/+ and Scn8a ^?3/+ heterozygous mutants display subtle motor deficits, reduced acoustic startle response, and are resistant to induced seizures, suggesting that these mutations reduce activity of the Scn8a channel protein, Na_v1.6. Heterozygous Scn8a ^Δ35/+ mutants show no alterations in motor function or acoustic startle response, but are resistant to induced seizures. Homozygous mutants from each line exhibit premature lethality and severe motor impairments, ranging from uncoordinated gait with tremor (Δ9 and ?3) to loss of hindlimb control (Δ35). Scn8a ^Δ9/Δ9 and Scn8a ^?3/?3 homozygous mutants also exhibit impaired nerve conduction velocity, while normal nerve conduction was observed in Scn8a ^Δ35/Δ35 homozygous mice. Our results suggest that hypomorphic mutations that reduce Na_v1.6 activity will likely result in different clinical phenotypes compared to null alleles. These three mouse lines represent a valuable opportunity to examine the phenotypic impacts of hypomorphic and null Scn8a mutations without the confound of strain‐specific differences.     

ChMCO1 of Cochliobolus heterostrophus is a new class of metallo-oxidase, playing an important role in DHN-melanization

A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallo-oxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (∆ChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that ∆ChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization.     

Amber mutants of Salmonella-phage P22 in genes engaged in the establishment of lysogeny

Summary Phage mutants were isolated with amber mutations in genes necessary for establishment of lysogeny. These mutants form turbid plaques on su ⁺ strain 527R1 and clear plaques of different types on LT2. According to complementation tests, fourteen mutants fall in the c ₂ gene, four in the c ₃ gene but no amber mutants were found belonging to the c ₁ gene. Pulse labelling experiments to follow DNA synthesis after phage infection were done with the mutants classified by complementation tests. Furthermore the labelling experiments demonstrated that the nonleaky c ₃ amber mutants displayed the same DNA synthesis pattern as c ₁ missense mutants. Since these c ₃ amber mutants complement missense c ₁ mutants it is concluded that the c ₃ and c ₁ genes must act together for the first transient repression of DNA synthesis, i.e., seven minutes after infection. It is suggested that clear plaque forming c ₁ amber mutants cannot be isolated because of polarity leading to defectivity of lysogenic as well as of lytic functions.The majority of the experiments presented are a part of the dissertation of H. D. Dopatka at the University of Göttingen.     

Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5 end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.     

Complementation of notabilis, an abscisic acid-deficient mutant of tomato: importance of sequence context and utility of partial complementation

The abscisic acid (ABA)‐deficient tomato mutant notabilis (not) is believed to be a null mutation in the gene LeNCED1, encoding a 9‐cis‐epoxycarotenoid dioxygenase involved in ABA biosynthesis. We have sequenced and analysed a 19 kb genomic clone containing LeNCED1 and 5.4 kb of its promoter. This clone was transferred to not homozygotes and several non‐wilty transformed plants were obtained. The basal ABA content, water relations, shoot and root growth, adventitious rooting, ethylene evolution and ability to accumulate ABA under water stress are described for two of these lines, not_comp.13 and not_comp.1. Partial complementation was observed for most parameters measured for not_comp.1. Full complementation was observed in not_comp.13 for all parameters measured in whole plants under well‐watered and water‐stressed conditions. These data provide further evidence that LeNCED1 is the wild‐type allele of the not mutant gene. However, not_comp.13 was unable to accumulate the wild‐type levels of ABA in rapidly dehydrated leaves, indicating that it too was only partially complemented. Since LeNCED1 is an environmentally regulated gene encoding a rate‐limiting enzyme, precise levels and patterns of gene expression may be needed to fully recreate wild‐type phenotype. The utility of partially complemented lines to study the role of ABA in plant responses to stress conditions, and in promoter analysis, is discussed.     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Characterization and fine mapping of the glabrous leaf and hull mutants (gl1) in rice (Oryza sativa L.)

The glabrous leaf and hull (gl1) mutants were isolated from M₂ generation of indica cultivar 93-11. These mutants produced smooth leaves and hairless glumes under normal growth conditions. By analyzing through scanning electron microscope, it was revealed that the leaf trichomes, including macro and micro hairs, were deficient in these mutants. Genetic analysis indicated that the mutation was controlled by a single recessive gene. Using nine SSR markers and one InDel marker, the gl1 gene was mapped between RM1200 and RM2010 at the short arm of chromosome 5, which was consistent with the mapping of gl1 in previous studies. To facilitate the map-based cloning of the gl1 gene, 12 new InDel markers were developed. A high-resolution genetic and physical map was constructed by using 1,396 mutant individuals of F₂ mapping population. Finally, the gl1 was fine mapped in 54-kb region containing 10 annotated genes. Cloning and sequencing of the target region from four gl1 mutants (gl1-1, gl1-2, gl1-3 and gl1-4) and four glabrous rice varieties (Jackson, Jefferson, Katy and Lemont) all showed that the same single point mutation (A→T) occurred in the 5′-untranslated region (UTR) of the locus Os05g0118900 (corresponding to the 3′-UTR of STAR2). RT-PCR analysis of the locus Os05g0118900 revealed that its mRNA expression level was normal in gl1 mutant. RNA secondary structure prediction showed that the single point mutation resulted in a striking RNA conformational change. These results suggest that the single point mutation is most likely responsible for the glabrous leaf and hull phenotypes in rice.     

Mutagenesis of a race-specific rust resistance gene in Antirrhinum majus using a transposon-tagging protocol

In a transposon tagging experiment, lines of Antirrhinum majus exhibiting both race-specific resistance (homozygous for the dominant R gene) to the rust pathogen Puccinia antirrhini and a high frequency of transposition were crossed with homozygous susceptible lines. From a screen of 11153 F₁ progeny, 15 individuals were detected showing susceptibility to rust race α. Six of these exhibited a susceptibility phenotype (classified as type B) not previously observed. A control experiment involving the same tagging strategy but employing lines that do not exhibit high rates of transposition did not yield any susceptible mutants from a screen of 6243 progeny. In experiments on the heritability and stability of the mutation, the six plants exhibiting susceptibility phenotype B produced progeny in which the R locus had reverted to an active form (i.e. some of the progeny were resistant), a classic characteristic of transposon-tagged plant genes. Reversion was shown to occur somatically, and its rate was temperature dependent. Inheritance studies showed that the mutations in two of the susceptible plants from the tagging protocol map at, or very close to, the race α-specific resistance gene. The results are consistent with the transposon tagging of a race-specific gene for rust resistance.