Characterization and Fine Mapping of a Necrotic Leaf Mutant in Maize (Zea mays L.)

Maize (Zea mays L.) is a commercially important crop. Its yield can be reduced by mutations in biosynthetic and degradative pathways that cause death. In this paper, we describe the necrotic leaf (nec-t) mutant, which was obtained from an inbred line, 81647. The nec-t mutant plants had yellow leaves with necrotic spots, reduced chlorophyll content, and the etiolated seedlings died under normal growth conditions. Transmission electron microscopy revealed scattered thylakoids, and reduced numbers of grana lamellae and chloroplasts per cell. Histochemical staining suggested that spot formation of nec-t leaves might be due to cell death. Genetic analysis showed that necrosis was caused by the mutation of a recessive locus. Using simple sequence repeat markers, the Nec-t gene was mapped between mmc0111 and bnlg2277 on the short arm of chromosome 2. A total of 1287 individuals with the mutant phenotype from a F₂ population were used for physical mapping. The Nec-t gene was located between markers T31 and H8 within a physical region of 131.7 kb.     

Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

Soil bacteria like Bacillus subtilis can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a B. subtilis mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic gudB^CR gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic gudB^CR allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic gudB^CR allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active gudB gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic gudB^CR allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient B. subtilis strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active gudB allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.     

A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

Using complementation tests and nucleotide sequencing, we showed that the rad58-4 mutation was an allele of the MRE11 gene and have renamed the mutation mre11-58. Two amino acid changes from the wild-type sequence were identified; one is located at a conserved site of a phosphodiesterase motif, and the other is a homologous amino acid change at a nonconserved site. Unlike mre11 null mutations, the mre11-58 mutation allowed meiosis-specific double-strand DNA breaks (DSBs) to form at recombination hot spots but failed to process those breaks. DSB ends of this mutant were resistant to lambda exonuclease treatment. These phenotypes are similar to those of rad50S mutants. In contrast to rad50S, however, mre11-58 was highly sensitive to methyl methanesulfonate treatment. DSB end processing induced by HO endonuclease was suppressed in both mre11-58 and the mre11 disruption mutant. We constructed a new mre11 mutant that contains only the phosphodiesterase motif mutation of the Mre11-58 protein and named it mre11-58S. This mutant showed the same phenotypes observed in mre11-58, suggesting that the phosphodiesterase consensus sequence is important for nucleolytic processing of DSB ends during both mitosis and meiosis.     

Excessive aluminium accumulation in the pea mutant E107 (brz)

E107 is a pleiotropic mutant of peaPisum sativum cv. ‘Sparkle’, characterized by forming few nodules and developing bronze necrotic spots on older leaves. The mutant accumulates Al and has symptoms typical of Al toxicity. The lateral roots of E107 are fewer (40%) and shorter (50%) than those of its parent. High concentrations of Al accumulate in E107 shoots (1000 mg kg^-1) and roots (3000 mg kg^-1), and three-week old E107 plants extrude 2.5 times more protons than ‘Sparkle’ plants of similar age. Al concentrations of the roots of the mutant and of its parent ‘Sparkle’ are similar for the first two weeks of growth. Thereafter they differ. In 2 week old plants Al continues to accumulate in excessive amounts in E107 primary and lateral roots whereas in ‘Sparkle’ roots, it reaches a plateau. In E107, Al is erratically distributed in the walls of root hairs and epidermal cells in both primary and lateral roots. Some of these cells have also Al in their nucleus.     

On the Enhanced Callose Deposition in Barley with ml-o Powdery Mildew Resistance Genes

Carborundum treatment of barley leaves induced a callose deposition which was detected as diffuse blotches in the epidermal cells of susceptible barleys and as deeply stained tracks along the scratches in barleys with the ml-o powdery mildew resistance gene. Subsequent inoculation with powdery mildew resulted in appositions that enlarged inversely to their size in the respective varieties when inoculated without carborundum treatment. Aphids sucking the leaves resulted in rows of callose containing spots along the anticlinal cell walls. The spots were larger in the ml-o mutant than in the mother variety. Callose was deposited in connection with the pleiotropic necrotic spotting in barleys with the ml-o gene. Modification of the necrotic spotting by crossing the ml-o gene into other gene backgrounds did not result in any change in the size of appositions upon inoculation with powdery mildew. Callose deposition was never observed in cells subsidiary to the guard cells, and the absence of callose in such cells is suggested to be the reason for the sporadic occurrence of powdery mildew colonies on barleys with the ml-o gene.     

Oxidative phosphorylation in Escherichia coli K-12: The genetic and biochemical characterisation of a strain carrying a mutation in the uncB gene

1. A mutant strain of Escherichia coli unable to grow with succinate as sole carbon source was isolated. This mutant was found to carry a mutation in a gene (designated uncB) mapping at about minute 73.5 on the E. coli chromosome and close to the uncA gene which is probably the structural gene for (Mg²⁺,Ca²⁺)-stimulated ATPase.2. The uncB401 allele was transduced into two other strains of E. coli and the transductants compared with the parent strains.3. Strains carrying the uncB401 allele have low aerobic growth yields when grown on limiting concentrations of glucose, but unlike mutations in the uncA gene, mutations in the uncB gene do not impair anaerobic growth on a glucose-mineral salts medium.4. Oxidase activities in membranes from the normal strains and strains carrying the uncB401 allele were similar.5. Measurement of P/O ratios indicated that a mutation in the uncB gene causes uncoupling of phosphorylation associated with electron transport with d-lactate as substrate.6. (Mg²⁺,Ca²⁺)-stimulated ATPase activities in the normal strains and in strains carrying the uncB401 allele are similar.7. Estimation of the energy-linked and non-energy-linked transhydrogenase activities in membrane preparations from both the normal and mutant strains indicated that the protein affected by a mutation in the uncB gene is essential for the functioning of the ATP-dependent energy-linked transhydrogenase.8. It is concluded that two proteins, specified by the uncA and uncB genes, are essential for phosphorylation coupled to d-lactate oxidation and also for the energy-linked transhydrogenase activity using ATP as the energy source.     

Genotoxic activities in vivo of cobaltous chloride and other metal chlorides as assayed in the Drosophila wing spot test

A series of metal chlorides were subjected to the wing spot test of Drosophila melanogaster. In the test, larvae trans-heterozygous for the wing-hair mutations mwh and flr were orally treated at the third instar stage with a test compound and the wings were inspected at the adult stage for spots expressing phenotypes of the markers. CoCl₂, MnCl₂, MoCl₃, NiCl₂ and ZnCl₂ were clearly effective in inducing spots with one or two mutant hairs (small spots). CoCl₂ was clearly effective in inducing spots with three or more mutant hairs (large spots) as well. CrCl₃, FeCl₂ and FeCl₃ were negative under the conditions used. Based on estimated frequencies of small spots induced at the LD₅₀, the genotoxic effectiveness of the positive metal salts were ranked in a sequence of CoCl₂ > ZnCl₂ > MoCl₃ > (MnCl₂, NiCl₂). Since CoCl₂ did not induce large spots in the wings of the mwh/TM3 flies with a suppressed ability of mitotic crossing-over, the large spots induced by this compound in the mwh/flr system were ascertained as mutant clones due to mitotic crossing-overs.     

An altered state of a specific en regulatory element induced in a maize tiller

There are numerous states of the regulatory element, Enhancer (En). With specific receptor alleles, such as a2m(r-pa-pu) or a2m(r), specific mutability patterns are expressed. One specific derivative En allele, En-v (En-variable), was originally identified with a coarse pattern of mutability with the a2m(r-pa-pu) allele and giving progeny with varied En expression (standard to reduced within an ear progeny). Derivatives of En-v were subsequently found on numerous occasions to give only a very reduced expression (fewer mutant spots) with the a2m(r-pa-pu) allele in the ears derived from the main stalk of the corn plant. When a comparison is made of the effect of this changed En-v state between tiller ears and main stalk ears of the same plant, the tiller ears show an increased level of En-v expression (coarse pattern), while the main-stalk ears continue to show the very reduced level of En-v expression (low frequency of very late variegation). This increased level of mutability of the tiller ears is maintained when transmitted through the main-stalk ear in the subsequent generation. These results indicate that heritable alterations of controlling elements can be produced by endogenous environmental factors present during normal plant development.     

Pathogenicity and taxonomy of Tenuignomonia styracis gen. et sp. nov., a new monotypic genus of Gnomoniaceae on Styrax obassia in Japan

Since 2010, an unknown fungus in the Gnomoniaceae has been found on overwintered leaves and petioles of Styrax obassia (Styracaceae) in Japan. This fungus is characterized by dark brown immersed or partially erumpent ascomata with long necks and fusiform to obovoid asci each with an acute or long tapering stipe. Each ascus bears eight fusiform to filiform ascospores. Our morphological observation and phylogenetic analyses based on the markers LSU, rpb2, and tef-1α indicated that this is a new monotypic genus in the Gnomoniaceae (Diaporthales), and Tenuignomonia styracis gen. et sp. nov. was descried herein. Members of the Gnomoniaceae are commonly isolated as endophytes, saprobes, and plant pathogens from a broad diversity of herbaceous, shade tree, and agriculturally significant plants. We thus carried out a pathogenicity test to determine if T. styracis is the causative agent of leaf blotch on S. obassia. One week after inoculation, this fungus produced small necrotic spots on the leaves and petioles, and all leaves having necrotic spots were abscised in a short time. We thus confirmed that this fungus has weak pathogenicity on S. obassia. This new species may promote early defoliation of S. obassia during the fall.     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Misleading herbivory in a tropical tree

The damage caused by herbivores can be confused with the drop of necrotic spots due to hypersensitive reactions. The negative impact of sessile herbivores on host plants is minimized by hypersensitive reactions that result in the death of attacked tissue. We reported a phenomenon that remarkably resembles herbivore damage, but is in fact a reaction to endophytic herbivores or pathogens. Although the damages on Tapirira guianensis leaves (Anacardiaceae) appear to be chew marks from herbivores, they are in fact dropped necrotic spots caused by gall-inducing insects. The phenomenon is widespread and challenges the view that gall-inducing insects inflict less damage on host plants, which was previously stated according to the rates of herbivory inflicted by free-feeding herbivores. The study highlights the need to reassess the past evaluations for some plant species, as they may have overestimated herbivory rates.     

Preparation of DNA-modified nanoparticles and preliminary study for colorimetric SNP analysis using their selective aggregations

DNA-modified nanospheres were prepared by anchoring amino-terminated oligodeoxynucleotides (ODNs) with carboxylates onto a colored polystyrene sphere surface through amido bonds. About 220 ODN molecules were immobilized onto a nanosphere 40 nm in diameter. Preliminary studies using the microspheres with 1 μm diameter reveal that the specificity of hybridization was retained after modification. Three kinds of differently colored (RGB, red/green/blue) nanospheres bearing unique ODNs on their surface were prepared for detecting the p53 gene. Each ODN is complementary to a different part in the 45mer sample that is a part of a conservative region of the p53 gene containing one of the hot spots. In a binary system using spheres R and G, the wild-type 45mer made the aggregates with yellow emission as the result of mixing both colors. The mutant 45mer containing one nucleotide displacement did not give such aggregates with distinct colors. The study of fluorescence resonance energy transfer (FRET) showed that spheres R and G directly contact each other in the aggregates with the wild type. The RGB ternary system gave aggregates with specific colors corresponding to the added ODN samples, wild type or mutant. In addition, in the presence of both samples, all of the spheres formed aggregates with white emission as a consequence of mixing three primary colors of light. This means that the present technique should allow us to conduct an allele analysis.     

Tissue-Specific Contributions of Paternally Expressed Gene 3 in Lactation and Maternal Care of Mus musculus

Paternally Expressed Gene 3 (Peg3) is an imprinted gene that controls milk letdown and maternal-caring behaviors. In this study, a conditional knockout allele has been developed in Mus musculus to further characterize these known functions of Peg3 in a tissue-specific manner. The mutant line was first crossed with a germline Cre. The progeny of this cross displayed growth retardation phenotypes. This is consistent with those seen in the previous mutant lines of Peg3, confirming the usefulness of the new mutant allele. The mutant line was subsequently crossed individually with MMTV- and Nkx2.1-Cre lines to test Peg3’s roles in the mammary gland and hypothalamus, respectively. According to the results, the milk letdown process was impaired in the nursing females with the Peg3 mutation in the mammary gland, but not in the hypothalamus. This suggests that Peg3’s roles in the milk letdown process are more critical in the mammary gland than in the hypothalamus. In contrast, one of the maternal-caring behaviors, nest-building, was interrupted in the females with the mutation in both MMTV- and Nkx2.1-driven lines. Overall, this is the first study to introduce a conditional knockout allele of Peg3 and to further dissect its contribution to mammalian reproduction in a tissue-specific manner.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F₂ progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.     

Clinical and neurocognitive outcome in symptomatic isovaleric acidemia

Background

Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.

Methods

To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.

Results

We found severely (??90:10) or moderately (??75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations.

Conclusions

Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.     

Dominant Mutation for Ethionine Resistance in Saccharomyces cerevisiae

An ethionine-resistant mutant of Saccharomyces cerevisiae has been investigated whose mutation (et^r2) confers resistance to the heterozygous diploid also containing the sensitive allele, et^s. The mutation is apparently specific for reversal of ethionine inhibition. The principal difference between the sensitive et^s strain and the mutant was the latter's inability to concentrate large intracellular quantities of adenosylethionine. Reduced incorporation of ethyl groups or ethionine in other cellular fractions of the mutant was also detected. The data show that the mutant has not lost the ability to form adenosylethionine. It is suggested that the mutant has an increased ability to hydrolyze this sulfonium compound after it has been synthesized. It is possible that some of the ethionine is detoxified before it can participate in protein or adenosylethionine synthesis. No mutant alteration in accumulation of ethionine from the medium was detected. In the presence of ethionine, the parental strain accumulated 25 times more adenosylethionine than did the mutant. However, with methionine, only twice as much adenosylmethionine was accumulated by the parental strain as by the mutant.     

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.)

Two Fix^? mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix^? (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix^? in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix^? line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).     

The Mutator-Related Cy Transposable Element of Zea Mays L. Behaves as a near-Mendelian Factor

The bz-rcy allele arose in a single gamete of the TEL (transposable-element laden) population, when the rcy receptor element inserted into the Bronze1 locus. This newly arisen receptor allele conditions a stable bronze kernel phenotype in the absence of the independently segregating regulatory element, Cy. In the presence of Cy, bz-rcy conditions fully colored spots on a bronze background. The spots represent clonal sectors arising from mutations of bz-rcy to Bz'. Although Cy exhibits genetic interactions with the Mutator system it differs from Mu-homologous elements in its near-Mendelian behavior which is in contrast to the non-Mendelian inheritance of Mutator and Mu-homologous elements. Evidence is presented which suggests that the timing and mode of Cy transposition differ from those of Mu1.