Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

正Dear Editor,CRISPR (clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants. The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA (sgRNA) incorporating a target sequence (Li et al., 2019). A Cas9/sgRNA complex re-     

A dynamic reversible RNA N6-methyladenosine modification: current status and perspectives

N⁶-methyladenosine (m⁶A), as the most abundant RNA epigenetic modifications, has been shown to play critical roles in various biological functions. Research about enzymes that can catalyze and remove m⁶A have revealed its comprehensive roles in messenger RNA (mRNA) metabolism and other physiological processes. The “readers” including YTH domain-containing proteins, hnRNPC, hnRNPG, hnRNPA2B1, IGF2BP1, IGF2BP2, and IGF2BP3, which can affect the fates of mRNA in an m⁶A-dependent manner. In this review, we focus on recent advances in the research of the m⁶A modifications, especially about the latest functions of its writers, erasers, readers in RNA metabolism, cancer, and lipid metabolism. In the end, we provide insights into the underlying molecular mechanisms of m⁶A modifications.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

FTO regulates adipogenesis by controlling cell cycle progression via m6A-YTHDF2 dependent mechanism

N⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes. Loss of m⁶A demethylase FTO increases m⁶A levels and inhibits adipogenesis of preadipocytes. However, its underlying mechanism remains elusive. Here, we demonstrated that silencing FTO inhibited adipogenesis of preadipocytes through impairing cell cycle progression at the early stage of adipogenesis. FTO knockdown markedly decreased the expression of CCNA2 and CDK2, crucial cell cycle regulators, leading to delayed entry of MDI-induced cells into G2 phase. Furthermore, the m⁶A levels of CCNA2 and CDK2 mRNA were significantly upregulated following FTO knockdown. m⁶A-binding protein YTHDF2 recognized and decayed methylated mRNAs of CCNA2 and CDK2, leading to decreased protein expression, thereby prolonging cell cycle progression and suppressing adipogenesis. Our work unravels that FTO regulates adipogenesis by controlling cell cycle progression in an m⁶A-YTHDF2 dependent manner, which provides insights into critical roles of m⁶A methylation in adipogenesis.     

Characterization and genetic mapping of a Photoperiod-sensitive dwarf 1 locus in rice (Oryza sativa L.)

Plant height is an important agronomic trait for crop architecture and yield. Most known factors determining plant height function in gibberellin or brassinosteroid biosynthesis or signal transduction. Here, we report a japonica rice (Oryza sativa ssp. japonica) dominant dwarf mutant, Photoperiod-sensitive dwarf 1 (Psd1). The Psd1 mutant showed impaired cell division and elongation, and a severe dwarf phenotype under long-day conditions, but nearly normal growth in short-day. The plant height of Psd1 mutant could not be rescued by gibberellin or brassinosteroid treatment. Genetic analysis with R₁ and F₂ populations determined that Psd1 phenotype was controlled by a single dominant locus. Linkage analysis with 101 tall F₂ plants grown in a long-day season, which were derived from a cross between Psd1 and an indica cultivar, located Psd1 locus on chromosome 1. Further fine-mapping with 1017 tall F₂ plants determined this locus on an 11.5-kb region. Sequencing analysis of this region detected a mutation site in a gene encoding a putative lipid transfer protein; the mutation produces a truncated C-terminus of the protein. This study establishes the genetic foundation for understanding the molecular mechanisms regulating plant cell division and elongation mediated by interaction between genetic and environmental factors.     

An improved method for pollen release from anther: a case study with mangroves

The estimation of pollen production is widely used in pollination and reproductive ecology, aerobiology and pollen-vegetation relationship studies. Pollen release is the key step in estimating pollen production and therefore, the technique used to release pollen is critical. Three methods, the drying method (DM), mechanical method (MM) and chemical method (CM), are used to determine the amount of pollen released from anthers. Few studies have compared the results obtained from each method, making it difficult to determine which method is most appropriate in a given situation. In this study, we compared existing methods with a new method that increases the amount of pollen released from anthers. Eight species of mangrove trees from the Neilingding-Futian National Nature Reserve were selected for study. We combined the MM and CM to produce a new method (the ultrasonic method, UM). To determine the best ultrasonic treatment time using the UM, different durations were tested and the various responses were analysed. The relationships between pollen rupture and the ultrasonic treatment time, pollen size, exine thickness and aperture size were analysed. Finally, four methods for pollen release from anthers were compared. The results indicated that the UM could be an efficient method to release pollen from the anther. To avoid pollen rupture, ultrasonic treatment times in the UM should be less than 30 s. The pollen rupture rate was significantly correlated with the ultrasonic treatment time (r = 0.618, p < 0.001) and the aperture size (r = 0.248, p = 0.036). This comparative experiment indicated that the UM can generate a greater pollen yield than other methods while causing less pollen rupture than the MM. Therefore, the UM may be suitable for more plant species and more accurate for pollen production estimates than the existing three methods, particularly when estimating pollen production in all community plants.     

A Novel Rice bHLH Transcription Factor, DTD, Acts Coordinately with TDR in Controlling Tapetum Function and Pollen Development

Dear Editor, Male reproductive development is an essential biological process for flowering plants and crucial for crop seed produc- tion. Formation of the male reproductive organ, the anther, involves a number of developmental events, including sta- men meristem specification, generation of sporogenous cells and their differentiation into microspore mother cells （MMCs）, meiosis, microspore （pollen） maturation, and polli- nation （Ma, 2005）. The formation of microspores and their development into mature pollen grains require cooperative interactions between gametophytic （microspores） and sporo- phytic （anther wall） cells, with the innermost cell layer, the tapetum, playing the most crucial role （Ma, 2005）. Tapetal cells underao decleneration bv Droclrammed cell death （PCD）.