A transcriptomic atlas of acute stress response to low pH in multiple Issatchenkia orientalis strains.

IMPORTANCE: Issatchenkia orientalis is a promising industrial chassis to produce biofuels and bioproducts due to its high tolerance to multiple environmental stresses such as low pH, heat, and other chemicals otherwise toxic for the most widely used microbes. Yet, little is known about specific mechanisms of such tolerance in this organism, hindering our ability to engineer this species to produce valuable biochemicals. Here, we report a comprehensive study of the mechanisms of acidic tolerance in this species via transcriptome profiling across variable pH for 12 different strains with different phenotypes. We found multiple regulatory mechanisms involved in tolerance to low pH in different strains of I. orientalis, marking potential targets for future gene editing and perturbation experiments.

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana.

BACKGROUND: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. RESULTS: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. CONCLUSIONS: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana.

Background: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. Results: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant seeds. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. Conclusions: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Alternatively, H2A.X may be involved in recruiting methyltransferases to those sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

Distinct regulatory pathways contribute to dynamic CHH methylation patterns in transposable elements throughout Arabidopsis embryogenesis.

CHH methylation (mCHH) increases gradually during embryogenesis across dicotyledonous plants, indicating conserved mechanisms of targeting and conferral. Although it is suggested that methylation increase during embryogenesis enhances transposable element silencing, the detailed epigenetic pathways underlying this process remain unclear. In Arabidopsis, mCHH is regulated by both small RNA-dependent DNA methylation (RdDM) and RNA-independent Chromomethylase 2 (CMT2) pathways. Here, we conducted DNA methylome profiling at five stages of Arabidopsis embryogenesis, and classified mCHH regions into groups based on their dependency on different methylation pathways. Our analysis revealed that the gradual increase in mCHH in embryos coincided with the expansion of small RNA expression and regional mCHH spreading to nearby sites at numerous loci. We identified distinct methylation dynamics in different groups of mCHH targets, which vary according to transposon length, location, and cytosine frequency. Finally, we highlight the characteristics of transposable element loci that are targeted by different mCHH machinery, showing that short, heterochromatic TEs with lower mCHG levels are enriched in loci that switch from CMT2 regulation in leaves, to RdDM regulation during embryogenesis. Our findings highlight the interplay between the length, location, and cytosine frequency of transposons and the mCHH machinery in modulating mCHH dynamics during embryogenesis.

Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate.

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

Divergence among rice cultivars reveals roles for transposition and epimutation in ongoing evolution of genomic imprinting.

Parent-of-origin-dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin-specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA-producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions-the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

A genetic toolbox for metabolic engineering of Issatchenkia orientalis.

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis.

The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-mediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1) subunit of the chromatin remodeler FACT (facilitates chromatin transactions), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in Arabidopsis endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in Arabidopsis We found that FACT colocalizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genome-wide. Our results show that the FACT-dependent DME targets are GC-rich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin.

Investigation of the Defect Structure of Congruent and Fe-Doped LiNbO3 Powders Synthesized by the Combustion Method.

Fe-doped LiNbO3 synthesized by the combustion method to seek new multiferroic materials exhibits room-temperature ferromagnetism, as reported in our previous work [1]. In this work, the defect structure of congruent and Fe-doped LiNbO3 (0.57-3.3 mol %) powders was investigated in detail by several methods. The molar ratio of [Li]/([Li]+[Nb]) was determined by the Curie temperature (Tc) via DSC. Two peaks of Tc were observed due to phase splitting [2], and the phase at lower Tc disappears as the Fe doping concentration increases. The coexistence of two different oxidation states of Fe ions in LiNbO3 was probed by XPS and UV-Vis spectroscopy. The Raman spectra exhibit displacements along the c axis of Li and Nb ions, and a deformation of the NbO6 framework owing to Fe doping. Several doping models were applied in the Rietveld refinement of powder X-ray diffraction collected by synchrotron radiation. The fitting by the Nb vacancy model leads to an improbably distorted structure of congruent LiNbO3. In Fe-doped LiNbO3, we conjecture that Li and Nb vacancies coexist in the lattice structure; Fe+2/Fe+3 ions are substituted for Li ions at the regular Li site and may push the anti-site NbLi ion back to the regular Nb site.

Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues.

Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells-the cell types that comprise pollen-with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations.

Effects of Rare Earth Metals on Steel Microstructures.

Rare earth metals are used in semiconductors, solar cells and catalysts. This review focuses on the background of oxide metallurgy technologies, the chemical and physical properties of rare earth (RE) metals, the background of oxide metallurgy, the functions of RE metals in steelmaking, and the influences of RE metals on steel microstructures. Future prospects for RE metal applications in steelmaking are also presented.

Comparative genomics of grass EST libraries reveals previously uncharacterized splicing events in crop plants.

BACKGROUND: Crop plants such as rice, maize and sorghum play economically-important roles as main sources of food, fuel, and animal feed. However, current genome annotations of crop plants still suffer false-positive predictions; a more comprehensive registry of alternative splicing (AS) events is also in demand. Comparative genomics of crop plants is largely unexplored. RESULTS: We performed a large-scale comparative analysis (ExonFinder) of the expressed sequence tag (EST) library from nine grass plants against three crop genomes (rice, maize, and sorghum) and identified 2,879 previously-unannotated exons (i.e., novel exons) in the three crops. We validated 81% of the tested exons by RT-PCR-sequencing, supporting the effectiveness of our in silico strategy. Evolutionary analysis reveals that the novel exons, comparing with their flanking annotated ones, are generally under weaker selection pressure at the protein level, but under stronger pressure at the RNA level, suggesting that most of the novel exons also represent novel alternatively spliced variants (ASVs). However, we also observed the consistency of evolutionary rates between certain novel exons and their flanking exons, which provided further evidence of their co-occurrence in the transcripts, suggesting that previously-annotated isoforms might be subject to erroneous predictions. Our validation showed that 54% of the tested genes expressed the newly-identified isoforms that contained the novel exons, rather than the previously-annotated isoforms that excluded them. The consistent results were steadily observed across cultivated (Oryza sativa and O. glaberrima) and wild (O. rufipogon and O. nivara) rice species, asserting the necessity of our curation of the crop genome annotations. Our comparative analyses also inferred the common ancestral transcriptome of grass plants and gain- and loss-of-ASV events. CONCLUSIONS: We have reannotated the rice, maize, and sorghum genomes, and showed that evolutionary rates might serve as an indicator for determining whether the identified exons were alternatively spliced. This study not only presents an effective in silico strategy for the improvement of plant annotations, but also provides further insights into the role of AS events in the evolution and domestication of crop plants. ExonFinder and the novel exons/ASVs identified are publicly accessible at http://exonfinder.sourceforge.net/ .

Electrically switchable cholesteric gratings based on slit electrodes.

An electrically switchable diffraction grating (ESDG) based on cholesteric liquid crystal (CLC) filled into the cell with slit electrodes is demonstrated in this study. On one hand, with low voltage, the ESDG has high second order diffraction efficiency because of the alternating planar and fingerprint textures. With high voltage, on the other hand, the ESDG has high first order diffraction efficiency because of the alternating planar and homeotropic textures. The first and second order diffraction efficiencies of ESDG are electrically swapped. The maximum diffraction efficiency of the ESDG is approximately 32% at each grating mode.

Local geometric and electronic structures of gasochromic VO(x) films.

VOx films were deposited by radio-frequency reactive magnetron sputtering from a vanadium target at room temperature. Local atomic and electronic structures of the films were then modified by thermal annealing. The oxidation state and structural and gasochromic properties of the films were elucidated by X-ray absorption spectroscopy. Analytical results indicate that the as-deposited VOx films were amorphous with mixed V(4+) and V(5+) valences. The amorphous VOx had a disordered and expanded lamellar structure resembling that of polymer-intercalated V2O5 gels. VOx films were crystallized into orthorhombic V2O5 at 300 °C, and the lamellar structure was eliminated at 400 °C. Additionally, the gasochromic reaction reduced the vanadium valence via intervalence transitions between V(5+) and V(3+). Moreover, removing the lamellar structure reduced the gasochromic rate, and the gasochromic reaction transformed the V2O5 crystalline phase irreversibly into an H1.43V2O5 phase. Based on the results of this study, amorphous VOx with a lamellar structure is recommended for use in H2 gas sensors.

The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin.

Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.