Potent pollen gene regulation by DNA glycosylases in maize.

Although DNA methylation primarily represses transposable elements (TEs) in plants, it also represses select endosperm and pollen genes. These genes, or their cis-regulatory elements, are methylated in plant body tissues but are demethylated by DNA glycosylases (DNGs) in endosperm and pollen, enabling their transcription. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes, whose expression is strongly decreased in double mutant pollen (124-fold decrease on average). These genes account for 11.1% of the wild-type pollen polyadenylated transcriptome, but they are silent or barely detectable in the plant body. They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their coding DNA sequence. Moreover, they are strongly enriched for predicted functions in cell wall modification. While some may support development of the pollen grain cell wall, expansins and pectinases in this set of genes suggest a function in cell wall loosening to support the rapid tip growth characteristic of pollen tubes as they carry the sperm cells through maternal apoplast and extracellular matrix of the pistil. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.

Haplotype-resolved genome assembly of Populus tremula × P. alba reveals aspen-specific megabase satellite DNA.

Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717-1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence-sensitive analyses. Here we report a telomere-to-telomere genome for this hybrid aspen with two chromosome-scale, haplotype-resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array-based and array-less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus) but PtaM147-like sequences occur in LTR-retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis-driven research to probe into the function of the abundant and aspen-specific PtaM147 satellite DNA.

Natural methylation epialleles correlate with gene expression in maize.

DNA methylation in plants is depleted from cis-regulatory elements in and near genes but is present in some gene bodies, including exons. Methylation in exons solely in the CG context is called gene body methylation (gbM). Methylation in exons in both CG and non-CG contexts is called TE-like methylation (teM). Assigning functions to both forms of methylation in genes has proven to be challenging. Toward that end, we utilized recent genome assemblies, gene annotations, transcription data, and methylome data to quantify common patterns of gene methylation and their relations to gene expression in maize. We found that gbM genes exist in a continuum of CG methylation levels without a clear demarcation between unmethylated genes and gbM genes. Analysis of expression levels across diverse maize stocks and tissues revealed a weak but highly significant positive correlation between gbM and gene expression except in endosperm. gbM epialleles were associated with an approximately 3% increase in steady-state expression level relative to unmethylated epialleles. In contrast to gbM genes, which were conserved and were broadly expressed across tissues, we found that teM genes, which make up about 12% of genes, are mainly silent, are poorly conserved, and exhibit evidence of annotation errors. We used these data to flag teM genes in the 26 NAM founder genome assemblies. While some teM genes are likely functional, these data suggest that the majority are not, and their inclusion can confound the interpretation of whole-genome studies.

Synthetic maize centromeres transmit chromosomes across generations.

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a tethering approach that activates functional maize centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient to organize functional kinetochores that caused chromosome breakage, releasing chromosome fragments that were passed through meiosis and into progeny. Several fragments formed independent neochromosomes with centromeres localized over the LexO repeat arrays. The new centromeres were self-sustaining and transmitted neochromosomes to subsequent generations in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

Genotypic-specific hormonal reprogramming and crosstalk are crucial for root growth and salt tolerance in bermudagrass (Cynodon dactylon).

Salt stress is one of the major abiotic factors limiting the productivity of bermudagrass (Cynodon dactylon). However, the role of hormonal reprogramming and crosstalk in regulating root growth and salt tolerance in bermudagrass was not reported. Here, we examined the physiological and hormonal responses of two contrasting bermudagrass genotypes; 'C43,' salt-tolerant 'C198' salt-sensitive. Under salt stress, 'C43' had better membrane stability and higher photosynthetic activity than the 'C198.' Salt stress promoted root growth and improved root/shoot ratio and root activity in 'C43,' but the root growth of 'C198' was inhibited by salt stress, leading to diminished root activity. The two bermudagrass genotypes also showed critical differences in hormonal responses, especially in the roots. The root contents of indole-3-acetic acid (IAA), cytokinin derivatives, such as trans-zeatin riboside (tZR) and dihydrozeatin riboside (DHZR) were increased in 'C43,' but decreased in 'C198' when exposed to salt stress. The root growth rate was positively correlated with the root IAA, tZR and DHZR, indicating their crucial role in root growth under salt stress. The expressions of TAA/YUCCA and CYP735A involved in IAA and tZR biosynthesis were induced by salt stress in 'C43,' but inhibited in 'C198,' leading to reduced hormone accumulations. Salt stress decreased the iP, tZ, and DHZ content in the roots of both genotypes, and no significant difference was observed between the two genotypes. Salt stress reduced the content of GA3 in both genotypes by inhibiting GA20ox and GA2ox genes, which could be attributed to the reduced shoot growth in both genotypes. The increased ABA level by salt stress was significantly higher in 'C198' than 'C43.' Furthermore, there were positive and negative correlations between different hormones and root growth, suggesting that root growth could be regulated by complex hormonal reprogramming and crosstalk. This study provides a foundation for understanding the underlying mechanisms of hormonal-mediated root growth and salt tolerance in bermudagrass.

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.

Intensification of sugar production by using Tween 80 to enhance metal-salt catalyzed pretreatment and enzymatic hydrolysis of sugarcane bagasse.

In this study, different metal-salt catalyzed pretreatment was presented to disorganize the obstinate structure by eliminating the majority of hemicellulose, fractional of lignin, and improve the enzymatic saccharification of sugarcane bagasse. With the accession of Tween 80 during enzymolysis, all metal-salt pretreated substrates presented higher glucose yields, especially for CuCl2. Furthermore, Tween 80 was added to the pretreatment, enhancing the elimination of hemicellulose and lignin, decreasing the degradation of sugars to inhibitors, and presenting superior performance on improving glucose yield. In addition, the maximum glucose yield of 88.0% was achieved by using Tween 80 concomitantly with AlCl3 pretreatment and enzymolysis. It was also found that adding Tween 80 during pretreatment or/and enzymolysis after 24 h could liberate the similar glucose without Tween 80 after 72 h. However, the enhancement of Tween 80 at 6 h was higher than that at 72 h.

Genetic Diversity and Population Structure of Phyllosticta citriasiana in China.

Phyllosticta citriasiana is the causal agent of citrus tan spot, an important pomelo disease in Asia. At present, there is little or no information on the epidemiology or population structure of P. citriasiana. By using simple sequence repeat markers, we analyzed 94 isolates from three pomelo production regions in southern and southeastern China. The analyses showed high genetic diversity in each of the three geographic populations. A STRUCTURE analysis revealed two genetic clusters among the 94 isolates; one geographic population was dominated by genotypes in one cluster, and the other two geographic populations were dominated by genotypes of the second cluster. P. citriasiana has a heterothallic mating system with two idiomorphs, MAT1-1 and MAT1-2. Analyses using mating type-specific primers revealed that both mating types were present in all three geographic populations, and in all three populations the mating type ratios were in equilibrium. Although the sexual stage of the fungus has not been discovered yet, analyses of allelic associations indicated evidence for sexual and asexual reproduction within and between populations. Despite the observed genetic differentiation between the three geographic populations, evidence for long-distance gene flow was found.

Genomic Sequencing of Phyllosticta citriasiana Provides Insight Into Its Conservation and Diversification With Two Closely Related Phyllosticta Species Associated With Citrus.

Phyllosticta capitalensis, Phyllosticta citricarpa, and Phyllosticta citriasiana are three very important Phyllosticta species associated with citrus. P. capitalensis is an endophyte fungus of citrus while P. citricarpa can cause black spot of citrus (e.g., oranges and mandarins). P. citriasiana was identified recently which is the causal agent of the pomelo tan spot. Here, we present the ∼34 Mb genome of P. citriasiana. The genome is organized in 92 contigs, encompassing 9202 predicted genes. Comparative genomic analyses with two other Phyllosticta species (P. citricarpa and P. capitalensis) associated with citrus was conducted to understand their evolutionary conservation and diversification. Pair-wise genome alignments revealed that these species are highly syntenic. All species encode similar numbers of CAZymes and secreted proteins. However, the molecular functions of the secretome showed that each species contains some enzymes with distinct activities. The three Phyllosticta species investigated shared a core set of 7261 protein families. P. capitalensis had the largest set of orphan genes (1991), in complete contrast to that of P. citriasiana (364) and P. citricarpa (262). Most of the orphan genes are functionally unknown, but they contain a certain number of species-specific secreted proteins. A total of 23 secondary metabolites biosynthesis clusters were identified in the three Phyllosticta species, 21 of them being highly conserved among these species while the remaining two showed whole cluster gain and loss polymorphisms or gene content polymorphisms. Taken together, our study reveals insights into the genetic mechanisms of host adaptation of three species of Phyllosticta associated with citrus and paves the way to identify effectors that function in infection of citrus plants.

How the Crosslinking Agent Influences the Thermal Stability of RTV Phenyl Silicone Rubber.

In this work, a thermal degradation mechanism of room temperature vulcanized (RTV) phenyl silicone rubber that was vulcanized by different crosslinking agents was discussed. Firstly, RTV phenyl silicone rubber samples were prepared by curing hydroxyl-terminated polymethyldiphenylsiloxane via three crosslinking agents, namely, tetraethoxysilane (TEOS), tetrapropoxysilane (TPOS), and polysilazane. Secondly, the ablation properties of RTV phenyl silicone rubber were studied by the muffle roaster test and FT-IR. Thirdly, thermal stability of the three samples was studied by thermogravimetric (TG) analysis. Finally, to explore the thermal degradation mechanism, the RTV phenyl silicone rubber vulcanized by different crosslinking agents were characterized by TG analysis-mass spectrum (TG-MS) and pyrolysis gas chromatogram-mass spectrum (pyGC-MS). Results showed that the thermal stability of RTV phenyl silicone rubber is related to the amount of residual Si⁻OH groups. The residual Si⁻OH groups initiated the polysiloxane chain degradation via an 'unzipping' mechanism.

Synthesis and anti-cancer activity of a glycosyl library of N-acetylglucosamine-bearing oleanolic acid.

N-Acetylglucosamine-bearing triterpenoid saponins (GNTS) were reported to be a unique type of saponins with potent anti-tumor activity. In order to study the structure-activity relationship of GNTS, 24 oleanolic acid saponins with (1 --> 3)-linked, (1 --> 4)-linked, (1 --> 6)-linked N-acetylglucosamine oligosaccharide residues were synthesized in a combinatorial and concise method. The cytotoxicity of these compounds toward the leukemia cell line HL-60 and the colorectal cancer cell line HT-29 could not be improved. Half maximal inhibition below 10 µM was achieved in one single case. The study revealed that the activity decreased following the order of 3' > 4' > 6' glycosyl modifications. GNTS that incorporated (D/L)-xylose and L-arabinose at positions 3' and 4' were more potent than those bearing other sugars.