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Reports on the measurement of low-intensity photon emissions from a metal-oxide-semiconductor capacitor biased with a voltage using the gated photon counting technique. Diagram for the photon counting experiment; Data on the synchronized photon count and displacement current.

Athyrium yokoscense is hypertolerant to cadmium (Cd) and can grow normally under a high Cd concentration despite Cd being a highly toxic heavy metal. To mitigate Cd stress in general plant species, Cd is promptly chelated with a thiol compound and is isolated into vacuoles. Generated active oxygen species (ROS) in the cytoplasm are removed by reduced glutathione. However, we found many differences in the countermeasures in A. yokoscense. Thiol compounds accumulated in the stele of the roots, although a long-term Cd exposure induced Cd accumulation in the aerial parts. Synchrotron radiation-based X-ray fluorescence (SR-XRF) analysis indicated that a large amount of Cd was localized in the cell walls of the roots. Overexpression of AyNramp5a, encoding a representative Fe and Mn transporter of A. yokoscense, increased both Cd uptake and Fe and Mn uptake in rice calli under the Cd exposure conditions. Organic acids are known to play a key role in reducing Cd availability to the plants by forming chelation and preventing its entry in free form into the roots. In A. yokoscense roots, Organic acids were abundantly detected. Investigating the chemical forms of the Cd molecules by X-ray absorption fine structure (XAFS) analysis detected many compounds with Cd-oxygen (Cd-O) binding in A. yokoscense roots, whereas in the aerial parts, the ratio of the compounds with Cd-sulfur (Cd-S) binding was increased. Together, our results imply that the strong Cd tolerance of A. yokoscense is an attribute of the following two mechanisms: Cd-O compound formation in the cell wall is a barrier to reduce Cd uptake into aerial parts. Thiol compounds in the region of root stele are involved in detoxication of Cd by formation of Cd-S compounds., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Cytoplasmic male sterility (CMS) is a mitochondrial-encoded trait that confers reproductive defects in males but not in females or any vegetative function. Why CMS is so often found in plants should be investigated from the viewpoint of mitochondrial phylogeny. Beta vulgaris, including the wild subspecies maritima and cultivated subspecies vulgaris (e.g., sugar beet), is known to be mitochondrially polymorphic, from which multiple CMS mitochondria have been found, but their evolutionary relationship has been obscure. We first refined the B. vulgaris reference mitochondrial genome to conduct a more accurate phylogenetic study. We identified mitochondrial single-nucleotide polymorphic sites from 600 B. vulgaris accessions. Principal component analysis, hierarchical clustering analysis, and creation of a phylogenetic tree consistently suggested that B. vulgaris mitochondria can be classified into several groups whose geographical distribution tends to be biased toward either the Atlantic or Mediterranean coasts. We examined the distribution of CMS-associated mitochondrial genes from Owen, E- and G-type CMS mitochondria. About one-third of cultivated beets had Owen-type CMS, which reflects the prevalence of using Owen-type CMS in hybrid breeding. Occurrence frequencies for each of the three CMS genes in wild beet were less than 4%. CMS genes were tightly associated with specific mitochondrial groups that are phylogenetically distinct, suggesting their independent origin. However, homologous sequences of the Owen type CMS gene occurred in several different mitochondrial groups, for which an intricate explanation is necessary. Whereas the origin of cultivated beet had been presumed to be Greece, we found an absence of Owen-type mitochondria in Greek accessions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kubota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Heteroplasmy, the coexistence of multiple mitochondrial DNA (mtDNA) sequences in a cell, is well documented in plants. Next-generation sequencing technology (NGS) has made it feasible to sequence entire genomes. Thus, NGS has the potential to detect heteroplasmy; however, the methods and pitfalls in heteroplasmy detection have not been fully investigated and identified. One obstacle for heteroplasmy detection is the sequence homology between mitochondrial-, plastid-, and nuclear DNA, of which the influence of nuclear DNA segments homologous to mtDNA (numt) need to be minimized. To detect heteroplasmy, we first excluded nuclear DNA sequences of sugar beet (Beta vulgaris) line EL10 from the sugar beet mtDNA sequence. NGS reads were obtained from single plants of sugar beet lines NK-195BRmm-O and NK-291BRmm-O and mapped to the unexcluded mtDNA regions. More than 1000 sites exhibited intra-individual polymorphism as detected by genome browsing analysis. We focused on a 309-bp region where 12 intra-individual polymorphic sites were closely linked to each other. Although the existence of DNA molecules having variant alleles at the 12 sites was confirmed by PCR amplification from NK-195BRmm-O and NK-291BRmm-O, these variants were not always called by six variant-calling programs, suggesting that these programs are inappropriate for intra-individual polymorphism detection. When we changed the nuclear DNA reference, a numt absent from EL10 was found to include the 309-bp region. Genetic segregation of an F2 population from NK-195BRmm-O x NK-291BRmm-O supported the numt origin of the variant alleles. Using four references, we found that numt detection exhibited reference dependency, and extreme polymorphism of numts exists among sugar beet lines. One of the identified numts absent from EL10 is also associated with another intra-individual polymorphic site in NK-195mm-O. Our data suggest that polymorphism among numts is unexpectedly high within sugar beets, leading to confusion about the true degree of heteroplasmy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Taniguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytoplasmic male sterility (CMS) is an increasingly important issue within the context of hybrid seed production. Its genetic framework is simple: S-cytoplasm for male sterility induction and dominant allele of the restorer-of-fertility gene (Rf) for suppression of S. However, breeders sometimes encounter a phenotype of CMS plants too complex to be explained via this simple model. The molecular basis of CMS provides clue to the mechanisms that underlie the expression of CMS. Mitochondria have been associated with S, and several unique ORFs to S-mitochondria are thought to be responsible for the induction of male sterility in various crops. Their functions are still the subject of debate, but they have been hypothesized to emit elements that trigger sterility. Rf suppresses the action of S by various mechanisms. Some Rfs, including those that encode the pentatricopeptide repeat (PPR) protein and other proteins, are now considered members of unique gene families that are specific to certain lineages. Additionally, they are thought to be complex loci in which several genes in a haplotype simultaneously counteract an S-cytoplasm and differences in the suite of genes in a haplotype can lead to multiple allelism including strong and weak Rf at phenotypic level. The stability of CMS is influenced by factors such as the environment, cytoplasm, and genetic background; the interaction of these factors is also important. In contrast, unstable CMS becomes inducible CMS if its expression can be controlled. CMS becomes environmentally sensitive in a genotype-dependent manner, suggesting the feasibility of controlling the expression of CMS., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Cytoplasmic male sterility in sugar beet becomes thermo-sensitive when combined with specific genotypes, potentially offering a means to environmentally control pollination by this trait. The stability of cytoplasmic male sterility expression in several genetic backgrounds was investigated in sugar beet (Beta vulgaris L.). Nine genetically heterogenous plants from open-pollinated varieties were crossed with a cytoplasmic male sterile line to obtain 266 F 1 plants. Based on marker analysis using a multiallelic DNA marker linked to restorer-of-fertility 1 (Rf1), we divided the F 1 plants into 15 genotypes. We evaluated the phenotypes of the F 1 plants under two environmental conditions: greenhouse rooms with or without daytime heating during the flowering season. Three phenotypic groups appeared: those consistently expressing male sterility, those consistently having restored pollen fertility, and those expressing male sterility in a thermo-sensitive manner. All plants in the consistently male sterile group inherited a specific Rf1 marker type named p4. We tested the potential for thermo-sensitive male sterile plants to serve as seed parents for hybrid seed production, and three genotypes were selected. Open pollination by a pollen parental line with a dominant trait of red-pigmented hypocotyls and leaf veins resulted in seed setting on thermo-sensitive male sterile plants, indicating that their female organs were functional. More than 99.9% of the progeny expressed the red pigmentation trait; hence, highly pure hybrids were obtained. We determined the nucleotide sequences of Rf1 from the three genotypes: One had a novel allele and two had known alleles, of which one was reported to have been selected previously as a non-restoring allele at a single U.S. breeding station but not at other stations in the U.S., or in Europe or Japan, suggesting environmental sensitivity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cytoplasmic male sterility (MS) in plants is caused by MS-inducing mitochondria, which have emerged frequently during plant evolution. Nuclear restorer-of-fertility (Rf)genes can suppress their cognate MS-inducing mitochondria. Whereas many Rfs encode a class of RNA-binding protein, the sugar beet (Caryophyllales) Rf encodes a protein resembling Oma1, which is involved in the quality control of mitochondria. In this study, we investigated the molecular evolution of Oma1 homologs in plants. We analyzed 37 plant genomes and concluded that a single copy is the ancestral state in Caryophyllales. Among the sugar beet Oma1 homologs, the orthologous copy is located in a syntenic region that is preserved in Arabidopsis thaliana. The sugar beet Rf is a complex locus consisting of a small Oma1 homolog family (RF-Oma1 family) unique to sugar beet. The gene arrangement in the vicinity of the locus is seen in some but not all Caryophyllalean plants and is absent from Ar. thaliana. This suggests a segmental duplication rather than a whole-genome duplication as the mechanism of RF-Oma1 evolution. Of thirty-seven positively selected codons in RF-Oma1, twenty-six of these sites are located in predicted transmembrane helices. Phylogenetic network analysis indicated that homologous recombination among the RF-Oma1 members played an important role to generate protein activity related to suppression. Together, our data illustrate how an evolutionarily young Rf has emerged from a lineage-specific paralog. Interestingly, several evolutionary features are shared with the RNA-binding protein type Rfs. Hence, the evolution of the sugar beet Rf is representative of Rf evolution in general., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production in many crops. Sugar beet CMS is associated with a unique mitochondrial protein named preSATP6 that forms a 250-kDa complex. Restorer-of-fertility 1 (Rf1) is a nuclear gene that suppresses CMS and is, hence, one of the targets of sugar beet breeding. Rf1 has dominant, semi-dominant and recessive alleles, suggesting that it may be a multi-allelic locus; however, the molecular basis for differences in genetic action is obscure. Molecular cloning of Rf1 revealed a gene (orf20) whose protein products produced in transgenics can bind with preSATP6 to generate a novel 200-kDa complex. The complex is also detected in fertility-restored anthers concomitant with a decrease in the amount of the 250-kDa complex. Molecular diversity of the Rf1 locus involves organizational diversity of a gene cluster composed of orf20-like genes (RF-Oma1s). We examined the possibility that members of the clustered RF-Oma1 in this locus could be associated with fertility restoration., Results: Six yet uncharacterized RF-Oma1s from dominant and recessive alleles were examined to determine whether they could generate the 200-kDa complex. Analyses of transgenic calli revealed that three RF-Oma1s from a dominant allele could generate the 200-kDa complex, suggesting that clustered RF-Oma1s in the dominant allele can participate in fertility restoration. None of the three copies from two recessive alleles was 200-kDa generative. The absence of this ability was confirmed by analyzing mitochondrial complexes in anthers of plants having these recessive alleles. Together with our previous data, we designed a set of PCR primers specific to the 200-kDa generative RF-Oma1s. The amount of mRNA measured by this primer set inversely correlated with the amount of the 250-kDa complex in anthers and positively correlated with the strength of the Rf1 alleles., Conclusions: Fertility restoration by sugar beet Rf1 can involve multiple RF-Oma1s clustered in the locus, implying that stacking 200-kDa generative copies in the locus strengthens the efficacy, whereas the absence of 200-kDa generative copies in the locus makes the allele recessive irrespective of the copy number. We propose that sugar beet Rf1 is a complex locus.

Athyrium yokoscense shows strong tolerance to cadmium exposure, even at levels that are many times greater than the toxic levels in ordinary plants. To determine the mechanism of Cd tolerance in A. yokoscense, we grew these plants under high Cd conditions and observed the tissue-specific accumulation of Cd and generation of reactive oxygen species, which is one of the major physiological responses to Cd stress. Fuchsin staining indicated the existence of a casparian strip in A. yokoscense roots, which may participate in Cd hypertolerance in A. yokoscense. Moreover, we performed RNA-seq of RNA samples from A. yokoscense plants treated with or without Cd exposure and obtained comprehensive RNA sequences as well as the Cd-responsive expression patterns of individual genes. Through de novo transcriptome assembly and gene expression analysis, we found that A. yokoscense showed normal features with no significant change in the expression levels of any transporter genes, even under high Cd exposure conditions. Our results demonstrate that A. yokoscense has an unusual mechanism that allows the invading Cd to partition into the distal roots, thus avoiding translocation of Cd into the xylem.

Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production. Although male sterility is caused by S cytoplasm (male-sterility inducing mitochondria), the action of S cytoplasm is suppressed by restorer-of-fertility ( Rf ), a nuclear gene. Hence, the genetics of Rf has attained particular interest among plant breeders. The genetic model posits Rf diversity in which an Rf specifically suppresses the cognate S cytoplasm. Molecular analysis of Rf loci in plants has identified various genes; however, pentatricopeptide repeat (PPR) protein (a specific type of RNA-binding protein) is so prominent as the Rf -gene product that Rf s have been categorized into two classes, PPR and non-PPR. In contrast, several shared features between PPR- and some non-PPR Rf s are apparent, suggesting the possibility of another grouping. Our present focus is to group Rf s by molecular genetic classes other than the presence of PPRs. We propose three categories that define partially overlapping groups of Rf s: association with post-transcriptional regulation of mitochondrial gene expression, resistance gene-like copy number variation at the locus, and lack of a direct link to S-orf (a mitochondrial ORF associated with CMS). These groups appear to reflect their own evolutionary background and their mechanism of conferring S cytoplasm specificity.

Restorer-of-fertility ( Rf ) is a suppressor of cytoplasmic male sterility (CMS), a mitochondrion-encoded trait that has been reported in many plant species. The occurrence of CMS is considered to be independent in each lineage; hence, the question of how Rf evolved was raised. Sugar beet Rf resembles Oma1 , a gene for quality control of the mitochondrial inner membrane. Oma1 homologues comprise a small gene family in the sugar beet genome, unlike Arabidopsis and other eukaryotes. The sugar beet sequence that best matched Arabidopsis atOma1 was named bvOma1 ; sugar beet Rf ( RF1-Oma1 ) was another member. During anther development, atOma1 mRNA was detected from the tetrad to the microspore stages, whereas bvOma1 mRNA was detected at the microspore stage and RF1-Oma1 mRNA was detected during the meiosis and tetrad stages. A transgenic study revealed that, whereas RF1-Oma1 can bind to a CMS-specific protein and alter the higher-order structure of the CMS-specific protein complex, neither bvOma1 nor atOma1 show such activity. We favour the hypothesis that an ancestral Oma1 gene duplicated to form a small gene family, and that one of the copies evolved and acquired a novel expression pattern and protein function as an Rf , i.e. RF1-Oma1 evolved via neofunctionalization., Competing Interests: There are no competing interests., (© 2019 The Authors.)

Key Message: The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele. Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.

Cytoplasmic male sterility (CMS) is a plant trait that involves interactions between nuclear- and mitochondrial genomes. In CMS, the nuclear restorer-of-fertility gene (Rf), a suppressor of male-sterility inducing mitochondria, is one of the best known genetic factors. Other unidentified genetic factors may exist but have not been well characterized. In sugar beet (Beta vulgaris L.), CMS is used for hybrid seed production, but few male-sterility inducing nuclear genotypes exist. Such genotypes could be introduced from a closely related plant such as leaf beet, but first the fertility restoring genotype of the related plant must be characterized. Here, we report the discovery of a Japanese leaf beet accession 'Fukkoku-ouba' that has both male-sterility inducing and fertility restoring genotypes. We crossed the leaf beet accession with a sugar beet CMS line, developed succeeding generations, and examined the segregation of two DNA markers that are linked to two sugar beet Rfs, Rf1 and Rf2. Only the Rf2 marker co-segregated with fertility restoration in every generation, implying that the Rf1 locus in leaf beet is occupied by a non-restoring allele. Fertility restoration was incomplete without a genetic factor closely linked to Rf1, leading to the assumption that the Rf1 locus encodes a modifier that cannot restore fertility by itself but perhaps strengthens another Rf. We sequenced the apparently non-restoring 'Fukkoku-ouba' rf1 gene-coding region and found that it closely resembles a restoring allele. The protein product demonstrated its potential to suppress CMS in transgenic suspension cells. In contrast, 'Fukkoku-ouba' rf1 transcript abundance was highly reduced compared to that of the restoring Rf1. Consistently, changes in protein complexes containing CMS-associated mitochondrial protein in anthers were very minor. Accordingly, we concluded that 'Fukkoku-ouba' rf1 is a hypomorph that acts as a non-restoring allele but has the potential to support another Rf, i.e. it is a modifier candidate., Competing Interests: The authors have declared that no competing interests exist.

Light-emitting diodes (LEDs) are an artificial light source used in closed-type plant factories and provide a promising solution for a year-round supply of green leafy vegetables, such as lettuce (Lactuca sativa L.). Obtaining high-quality seedlings using controlled irradiation from LEDs is critical, as the seedling health affects the growth and yield of leaf lettuce after transplantation. Because key molecular pathways underlying plant responses to a specific light quality and intensity remain poorly characterised, we used a multi-omics-based approach to evaluate the metabolic and transcriptional reprogramming of leaf lettuce seedlings grown under narrow-band LED lighting. Four types of monochromatic LEDs (one blue, two green and one red) and white fluorescent light (control) were used at low and high intensities (100 and 300 μmol·m -2 ·s -1 , respectively). Multi-platform mass spectrometry-based metabolomics and RNA-Seq were used to determine changes in the metabolome and transcriptome of lettuce plants in response to different light qualities and intensities. Metabolic pathway analysis revealed distinct regulatory mechanisms involved in flavonoid and phenylpropanoid biosynthetic pathways under blue and green wavelengths. Taken together, these data suggest that the energy transmitted by green light is effective in creating a balance between biomass production and the production of secondary metabolites involved in plant defence.

First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log 10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal.

Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.

Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt., (© 2013 John Wiley & Sons Ltd.)

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer - of - fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After Hin dIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.

Strontium chloride 89 (89Sr) is used as a systemic radiopharmaceutical therapy for the palliation of pain in patients with metastatic bone cancer. A 64-year-old man had previously undergone an operation to resect his right upper lobe of lung and sixth rib. He was diagnosed with lung cancer (large cell carcinoma, pT3N0M0, stage IIB). Three months later, he was treated with chemoradiotherapy for local recurrence. Ten months later, he could not sit up due to severe pain of the left ilium, although he had been treated with opiate analgesics. Fourteen months later, his hospital stay was prolonged and he was treated with 89Sr. One week after injection, the pain was almost completed relieved. Two weeks after injection, morphine infusion was stopped and a reduced dose of a fentanyl patch was used. He was also able to eat meals. Three weeks after injection, he started rehabilitation. Two months after the injection of 89Sr, he could return home from the hospital. Adverse events included grade 2 leukopenia, neutropenia and thrombocytopenia. These peaked 2 months after injection.

Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

During the postpartum period there is a high incidence of mastitis in dairy cows. The reason for this increased risk of mastitis still remains unclear. Since leukocytes in colostrum have an important role in preventing the onset of mastitis, we investigated the leukocyte populations, which express CD4, CD8, CD14, CD21 or WC1, in colostrum as well as in blood obtained from 14 Holstein cows. Eight cows developed mastitis within a week after calving and the other 6 remained healthy. The percentage of CD14+ cells in colostrum was significantly lower in mastitic cows than in healthy cows. There were no significant differences in other marker positive cells either in the colostrum or in the blood. The CD14+ cells in colostrum play an important role of defense against invading microorganisms in the mammary glands. Our results suggested that the lower percentage of CD14+ cells in colostrum might predict the incidence of mastitis in the following period.

Background: Recently, serum beta2-microglobulin, an endogenous marker for renal function, has been shown to be an independent predictor of mortality in older adults. However, the prognostic role of beta2-microglobulin in heart failure has not been elucidated., Methods: We prospectively evaluated serum beta2-microglobulin and creatinine concentrations, creatinine-based renal parameters (estimated glomerular filtration rate and creatinine clearance), and echocardiographic data in 131 patients with acute heart failure and creatinine concentrations < or =3.0mg/dL admitted to our hospitals., Results: During 2.3+/-1.3 years, 42 patients died of cardiovascular causes and 12 died of noncardiac causes. Cardiovascular events were observed in 63 patients: 53 were readmitted due to worsening heart failure, 5 readmitted for cerebral embolism, and 5 died from sudden cardiac death. According to multivariate stepwise Cox proportional hazard analysis, higher baseline serum beta2-microglobulin concentrations (X(2)=16, p<0.0001), previous congestive heart failure (X(2)=11, p<0.001), presence of chronic obstructive pulmonary disease (X(2)=8, p<0.01), and lower diastolic blood pressure (X(2)=6, p<0.05) were independent predictors of increased cardiovascular events. Also, higher baseline serum beta2-microglobulin (X(2)=20, p<0.0001), lower systolic blood pressure (X(2)=11, p<0.001), higher relative left ventricular wall thickness (X(2)=6, p<0.05), and lower body mass index (X(2)=5, p<0.05) were independent predictors of increased cardiac mortality. The adjusted hazard ratio for cardiovascular events increased with baseline serum beta2-microglobulin above 2.1 mg/L: 2.9 with beta2-microglobulin of 2.2-2.6 mg/L (95%CI 1.2-6.9, p<0.05), 2.9 with beta2-microglobulin of 2.7-3.9 mg/L (95%CI 1.2-7.2, p<0.05), and 4.7 with beta2-microglobulin of > or =4.0 mg/L (95%CI 2.0-11, p<0.001)., Conclusions: Higher baseline serum beta2-microglobulin concentration could be a promising risk marker in acute heart failure patients with creatinine < or =3.0 mg/dL., (2009 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Extensive genome rearrangement is one of the major mechanisms of angiosperm mitochondrial evolution. As a by-product, some angiosperm mitochondrial genes exhibit divergent organization, but not all of these genes have been fully characterized. Sugar beet ccmC, which plays an important role in cytochrome c maturation, harbors a unique extended NH(2) terminal region of 277 amino acid residues (N-extension) instead of a conserved translational initiation codon. The 5' termini of two major RNA species were determined by primer extension analysis, which revealed that the larger transcript covered the entire N-extension. Nucleotide sequencing of the cDNA revealed that a total of 31 C-to-U RNA editing events occurred in the N-extension and the ccmC-homologous region (ccmC-core region), resulting in improvement of amino acid sequence conservation. Antiserum was raised against a synthetic peptide corresponding to the ccmC-core region and was used for protein gel blot analysis of sugar beet and radish mitochondrial proteins. The detected 29.5-kDa signal band is shared by sugar beet and radish. Two additional larger signal bands are exclusively detected from sugar beet. The largest signal band is also detected by anti-N-extension antiserum. Our results indicate that sugar beet ccmC is translated as a long precursor with N-extension.

Purpose: We estimated the coronary artery wall using maximum intensity fusion (MIF) of whole-heart magnetic resonance (MR) angiography (WHCA) and water suppression-spectral presaturation with inversion recovery (WS-SPIR) 3D T(1)-weighted turbo field echo (3DT(1) TFE)., Methods: We created a phantom using a wall of plastic bottles varied with plastic tapes measuring 0.4 to 3.0 mm thick (0-14 sheets) by vernier caliper and compared widths with those on profile curves. In 3 patients, to clarify the capacity to visualize the coronary wall in vulnerable plaque, we acquired WS-SPIR 3D T(1) TFE and WS-spectral attenuation with inversion recovery (SPAIR) (inversion time [TI] 400 ms) 3D T(1) TFE images of carotid vulnerable plaque; also termed "lipid-rich plaque," vulnerable plaque is considered to be visualized in high intensity. We utilized the same geometric parameters and rest period on WHCA as for WS-SPIR 3D T(1) TFE. We obtained MIF of WHCA and WS-SPIR 3D T(1) TFE and measured thickness of the right coronary artery (RCA) wall on the profile curve in 18 cases., Results: The widths of the dip of the lower third of the bottom to head on the profile curve were consistent with actual measurement at 1-2 mm, the usual coronary artery wall thickness. Carotid plaques of high intensity by T(1)-weighted black-blood (T(1)BB) and T(2)-weighted BB (T(2)BB) methods showed high intensity on WS-SPAIR (TI 400 ms) 3D T(1) TFE and low intensity on WS-SPIR 3D T(1) TFE. With or without vulnerable plaque in the coronary artery wall, MIF of WHCA and WS-SPIR 3D T(1) TFE reflected the coronary artery wall. We obtained bands of low intensity in MIF between epicardial fat of WS-SPIR 3D T(1) TFE and coronary artery lumen of WHCA all but mid RCA in all 18 cases. We were unable to detect mid RCA in 5 cases. The outline of the obstructed mid RCA in 1 case was clear in WS-SPIR 3D T(1) TFE. The higher velocity of RCA movement caused blurring in another 4 cases in both WHCA and WS-SPIR 3D T(1) TFE. Those wall thickness of proximal or mid RCA averaged 1.3+/-0.2 mm., Conclusion: Bands of low intensity between epicardial fat and coronary artery lumen on MIF of WHCA and WS-SPIR 3D T(1) TFE can reflect the coronary artery wall.

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.

Taurine is known to lower blood pressure in essential hypertension and some experimental hypertensive models. Taurine has also been reported to activate aldehyde dehydrogenase and to inhibit the elevation of plasma acetaldehyde concentration after ethanol intake. Because acetaldehyde, the first metabolite of ethanol, is suspected to be responsible for many adverse effects of alcohol consumption, we examined the effect of taurine supplementation on ethanol-induced hypertension and abnormalities in the intracellular cation metabolism in Witar-Kyoto rats. In Study 1, systolic blood pressure and intraplatelet free calcium were significantly higher in rats who received 15% ethanol in drinking water than in control rats. Oral taurine supplementation (1% taurine and 15% ethanol in drinking water) completely prevented the development of ethanol-induced hypertension. Intraerythrocyte sodium and intraplatelet free calcium were significantly decreased in taurine-supplemented rats as compared with rats who received 15% ethanol only. In Study 2, hemoglobin-associated acetaldehyde (HbAA) was measured as a marker of protein-bound acetaldehyde. HbAA was significantly elevated in rats who received 5% ethanol in drinking water as compared with control rats. Taurine supplementation (1% taurine and 5% ethanol in drinking water) significantly decreased HbAA. Our findings suggest that the oral supplementation of taurine prevents ethanol-induced hypertension by decreasing protein bound acetaldehyde and altering the cation handling by the membrane.

A 3 year-old female Japanese Black cattle was diagnosed as diabetes mellitus (DM). Hyperglycemia (295 mg/dl), increase of serum fructosamine (487 micromol/l), elevated glycosylated hemoglobin A1 (GHbA1; 10.9%), low concentration of serum insulin (< 1.0 microU/ ml), increased serum glucagon (399 pg/ml), and glucose intolerance (glucose disappearance rate; k=0.53) were noted. On the histopathologic findings in pancreas, insulitis with infiltration of mononuclear cells was found. This case suggests that serum fructosamine and GHbA1 are available parameters for understanding of pathophysiological conditions of bovine DM.