A Novel 3-O-rhamnoside: 2″-O-xylosyltransferase Responsible for Terminal Modification of Prenylflavonol Glycosides in Epimedium pubescens Maxim.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest Km value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.

Genome-Wide Analysis and Expression Profiling of Glutathione Reductase Gene Family in Oat (Avena sativa) Indicate Their Responses to Abiotic Stress during Seed Imbibition.

Abiotic stress disturbs plant cellular redox homeostasis, inhibiting seed germination and plant growth. This is a crucial limitation to crop yield. Glutathione reductase (GR) is an important component of the ascorbate-glutathione (AsA-GSH) cycle which is involved in multiple plant metabolic processes. In the present study, GRs in A. sativa (AsGRs) were selected to explore their molecular characterization, phylogenetic relationship, and RNA expression changes during seed imbibition under abiotic stress. Seven AsGR genes were identified and mapped on six chromosomes of A, C, and D subgenomes. Phylogenetic analysis and subcellular localization of AsGR proteins divided them into two sub-families, AsGR1 and AsGR2, which were predicted to be mainly located in cytoplasm, mitochondrion, and chloroplast. Cis-elements relevant to stress and hormone responses are distributed in promoter regions of AsGRs. Tissue-specific expression profiling showed that AsGR1 genes were highly expressed in roots, leaves, and seeds, while AsGR2 genes were highly expressed in leaves and seeds. Both AsGR1 and AsGR2 genes showed a decreasing-increasing expression trend during seed germination under non-stress conditions. In addition, their responses to drought, salt, cold, copper, H2O2, and ageing treatments were quite different during seed imbibition. Among the seven AsGR genes, AsGR1-A, AsGR1-C, AsGR2-A, and AsGR2-D responded more significantly, especially under drought, ageing, and H2O2 stress. This study has laid the ground for the functional characterization of GR and the improvement of oat stress tolerance and seed vigor.

Dynamic Responses of Antioxidant and Glyoxalase Systems to Seed Aging Based on Full-Length Transcriptome in Oat (Avena sativa L.).

Seed aging is a major challenge for food security, agronomic production, and germplasm conservation, and reactive oxygen species (ROS) and methylglyoxal (MG) are highly involved in the aging process. However, the regulatory mechanisms controlling the abundance of ROS and MG are not well characterized. To characterize dynamic response of antioxidant and glyoxalase systems during seed aging, oat (Avena sativa L.) aged seeds with a range of germination percentages were used to explore physiological parameters, biochemical parameters and relevant gene expression. A reference transcriptome based on PacBio sequencing generated 67,184 non-redundant full-length transcripts, with 59,050 annotated. Subsequently, eleven seed samples were used to investigate the dynamic response of respiration, ROS and MG accumulation, antioxidant enzymes and glyoxalase activity, and associated genes expression. The 48 indicators with high correlation coefficients were divided into six major response patterns, and were used for placing eleven seed samples into four groups, i.e., non-aged (Group N), higher vigor (Group H), medium vigor (Group M), and lower vigor (Group L). Finally, we proposed a putative model for aging response and self-detoxification mechanisms based on the four groups representing different aging levels. In addition, the outcomes of the study suggested the dysfunction of antioxidant and glyoxalase system, and the accumulation of ROS and MG definitely contribute to oat seed aging.

Unveiling the Complexity of Red Clover (Trifolium pratense L.) Transcriptome and Transcriptional Regulation of Isoflavonoid Biosynthesis Using Integrated Long- and Short-Read RNAseq.

Red clover (Trifolium pratense L.) is used as forage and contains a high level of isoflavonoids. Although isoflavonoids in red clover were discovered a long time ago, the transcriptional regulation of isoflavonoid biosynthesis is virtually unknown because of the lack of accurate and comprehensive characterization of the transcriptome. Here, we used a combination of long-read (PacBio Iso-Seq) and short-read (Illumina) RNAseq sequencing to develop a more comprehensive full-length transcriptome in four tissues (root, stem, leaf, and flower) and to identify transcription factors possibly involved in isoflavonoid biosynthesis in red clover. Overall, we obtained 50,922 isoforms, including 19,860 known genes and 2817 novel isoforms based on the annotation of RefGen Tp_v2.0. We also found 1843 long non-coding RNAs, 1625 fusion genes, and 34,612 alternatively spliced events, with some transcript isoforms validated experimentally. A total of 16,734 differentially expressed genes were identified in the four tissues, including 43 isoflavonoid-biosynthesis-related genes, such as stem-specific expressed TpPAL, TpC4H, and Tp4CL and root-specific expressed TpCHS, TpCHI1, and TpIFS. Further, weighted gene co-expression network analysis and a targeted compound assay were combined to investigate the association between the isoflavonoid content and the transcription factors expression in the four tissues. Twelve transcription factors were identified as key genes for isoflavonoid biosynthesis. Among these transcription factors, the overexpression of TpMYB30 or TpRSM1-2 significantly increased the isoflavonoid content in tobacco. In particular, the glycitin was increased by 50-100 times in the plants overexpressing TpRSM1-2, in comparison to that in the WT plants. Our study provides a comprehensive and accurate annotation of the red clover transcriptome and candidate genes to improve isoflavonoid biosynthesis and accelerate research into molecular breeding in red clover or other crops.

Transcriptomic analysis reveals the changes of energy production and AsA-GSH cycle in oat embryos during seed ageing.

Deterioration during seed storage generally causes seed vigour declining. However, the mechanism of deterioration occurred still not clear. Seeds and embryos of oat (Avena sativa L.) were selected to analyze the relation of physiological and metabolic reactions with DEGs by using RNA-seq. Oat seed vigour declined during seeds aged 0 day (CK), 16 days (CD16) and 32 days (CD32). The changes of MDA and H2O2 contents, antioxidant enzymes activities of APX, DHAR, MDHAR and GR related with AsA-GSH cycle in embryos illustrated that seed vigour declined to the minimum at CD32. Transcriptomic analysis showed a total of 11335 and 8274 DEGs were identified at CD16 and CD32 compared with CK respectively, of which 4070 were overlapped. When seed vigour declined to the moderate level (CD16), the accumulation of H2O2 caused by the inhibition of complex I in ETC could be alleviated with AsA-GSH cycle. RNA-seq and qRT-PCR results both showed alternative oxidase in alternate respiratory pathway was upregulated which would maintain seed respiration. However, as seed vigour was at the lowest level (CD32), blocked ETC caused by down-regulation of complex III, including Ubiquinol-cytochrome C reductase complex 14kD subunit and Ubiquinol-cytochrome C reductase, UQCRX/QCR9 like, were more seriously and H2O2 scavenging was limited by the inactive AsA-GSH cycle. It could be suggested that the function of AsA-GSH would play a key role for regulating the physiological responses of ETC in embryos during seed ageing. These results would provide an insight into embryo for the transcriptomic information during oat seed ageing.

An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines.

BACKGROUND: DNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance. RESULTS: We here describe a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells. CONCLUSION: The MMSDK method will be a valuable tool to increase the current knowledge of genome wide DNA methylation profiles.