Enhancing rutin accumulation in Tartary buckwheat through a novel flavonoid transporter protein FtABCC2.

Tartary buckwheat (Fagopyrum tataricum) is an annual coarse cereal from the Polygonaceae family, known for its high content of flavonoid compounds, particularly rutin. But so far, the mechanisms of the flavonoid transport and storage in Tartary buckwheat (TB) remain largely unexplored. This study focuses on ATP-binding cassette transporters subfamily C (ABCC) members, which are crucial for the biosynthesis and transport of flavonoids in plants. The evolutionary and expression pattern analyses of the ABCC genes in TB identified an ABCC protein gene, FtABCC2, that is highly correlated with rutin synthesis. Subcellular localization analysis revealed that FtABCC2 protein is specifically localized to the vacuole membrane. Heterologous expression of FtABCC2 in Saccharomyces cerevisiae confirmed that its transport ability of flavonoid glycosides such as rutin and isoquercetin, but not the aglycones such as quercetin and dihydroquercetin. Overexpression of FtABCC2 in TB hairy root lines resulted in a significant increase in total flavonoid and rutin content (P < 0.01). Analysis of the FtABCC2 promoter revealed potential cis-acting elements responsive to hormones, cold stress, mechanical injury and light stress. Overall, this study demonstrates that FtABCC2 can efficiently facilitate the transport of rutin into vacuoles, thereby enhancing flavonoids accumulation. These findings suggest that FtABCC2 is a promising candidate for molecular-assisted breeding aimed at developing high-flavonoid TB varieties.

Regulatory Module FtMYB5/6-FtGBF1-FtUFGT163 Promotes Rutin Biosynthesis in Tartary Buckwheat.

Tartary buckwheat is highly valued for its abundant rutin (quercetin 3-O-rutinoside). As a flavonoid glycoside, rutin is synthesized with the crucial involvement of UDP-dependent glycosyltransferases (UGTs). However, the functions and transcriptional regulation of the UGT-encoded genes remain poorly understood. This study identified a key gene, FtUFGT163, potentially encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase in Tartary buckwheat through omics analysis and molecular docking methods. The recombinant FtUFGT163 expressed in Escherichia coli demonstrated the capacity to glycosylate isoquercetin into rutin. Overexpression of FtUFGT163 significantly enhanced the rutin content in Tartary buckwheat. Further investigation identified a novel bZIP transcription factor, FtGBF1, that enhances FtUFGT163 expression by binding to the G-box element within its promoter, thereby augmenting rutin biosynthesis. Additional molecular biology experiments indicated that the specific positive regulator of rutin, FtMYB5/6, could directly activate the FtGBF1 promoter. Collectively, this study elucidates a novel regulatory module, termed "FtMYB5/6-FtGBF1-FtUFGT163", which effectively coordinates the biosynthesis of rutin in Tartary buckwheat, offering insights into the genetic enhancement of nutraceutical components in crops.

Palladium-catalysed aryl/monofluoroalkylation of allenamides: access to fluoroalkyl indoles and isoquinolones.

A palladium catalysed construction of fluoroalkyl indoles and isoquinolones through aryl/monofluoroalkylation of allenamides has been developed. Monofluoromethyl-substituted heterocycles could be accessed under mild conditions with broad functional group tolerance. In addition, indole-oxindole bisheterocyclic scaffolds bearing a fluorine atom were successfully synthesized with 3-fluoro-oxindole as the nucleophile by applying this method.

Tartary buckwheat rutin: Accumulation, metabolic pathways, regulation mechanisms, and biofortification strategies.

Rutin is a significant flavonoid with strong antioxidant property and various therapeutic effects. It plays a crucial role in disease prevention and human health maintenance, especially in anti-inflammatory, antidiabetic, hepatoprotective and cardiovascular effects. While many plants can synthesize and accumulate rutin, tartary buckwheat is the only food crop possessing high levels of rutin. At present, the rutin content (RC) is regarded as the key index for evaluating the nutritional quality of tartary buckwheat. Consequently, rutin has become the focus for tartary buckwheat breeders and has made considerable progress. Here, we summarize research on the rutin in tartary buckwheat in the past two decades, including its accumulation, biosynthesis and breakdown pathways, and regulatory mechanisms. Furthermore, we propose several strategies to increase the RC in tartary buckwheat seeds based on current knowledge. This review aims to provide valuable references for elevating the quality of tartary buckwheat in the future.

Three-Component Synthesis of Multiple Functionalized Allenes via Copper/Photoredox Dual Catalyzed 1,4-Alkylcyanation of 1,3-Enynes.

Efficient access to multiple functionalized allenes via a three component 1,4-alkylcyanation of enynes with cyclic alcohol derivatives in the presence of trimethylsilyl cyanide (TMSCN) under copper/photoredox dual catalysis has been developed. Both easily transformable aldehyde and cyano groups were introduced to tetra-substituted allenes through light-induced C-C bond cleavage of cyclic butanol and pentanol derivatives. The reactions proceeded smoothly under mild conditions with broad functional groups tolerance.

Electrophile-Controlled Regiodivergent Palladium-Catalyzed Imidoylative Spirocyclization of Cyclic Alkenes.

An intermolecular controllable Pd-catalyzed spirocyclization of isocyano cycloalkenes has been developed, offering efficient and selective approaches toward spirocyclic hydropyrrole scaffolds. 2-Azaspiro-1,7-dienes could be obtained through a "chain-walking" process with aryl/vinyl iodides as electrophiles, while the normal Heck product 2-azaspiro-1,6-dienes were selectively generated when aryl triflates were used as the coupling partner of isocyanides. Mechanistic studies suggested that the counteranion of the Pd(II) intermediate played a crucial role in the regioselectivity control. Dihydropyrrole-fused 5,6,7-membered spirocycles were switchably accessed under mild conditions with wide functional group tolerance.

Accumulation of the bitter substance quercetin mediated by the overexpression of a novel seed-specific gene FtRDE2 in Tartary buckwheat.

Tartary buckwheat (Fagopyrum tataricum) is frequently employed as a resource to develop health foods, owing to its abundant flavonoids such as rutin. However, the consumption of Tartary buckwheat (TB) is limited in food products due to the strong bitterness induced by the hydrolysis of rutin into quercetin. This transformation is facilitated by the degrading enzyme (RDE). While multiple RDE isoenzymes exist in TB, the superior coding gene of FtRDEs has not been fully explored, which hinders the breeding of TB varieties with minimal bitterness. Here, we found that FtRDE2 is the most abundant enzyme in RDE crude extracts, and its corresponding gene is specifically expressed in TB seeds. Results showed that FtRDE2 has strong rutin hydrolysis activity. Overexpression of FtRDE2 not only significantly promoted rutin hydrolysis and quercetin accumulation but also dramatically upregulated genes involved in the early phase of flavonoid synthesis (FtPAL1、FtC4H1、Ft4CL1, FtCHI1) and anthocyanin metabolism (FtDFR1). These findings elucidate the role of FtRDE2, emphasizing it as an endogenous factor contributing to the bitterness in TB and its involvement in the metabolic regulatory network. Moreover, correlation analysis revealed a positive relationship between the catalytic activity of RDE extracts and the expression level of FtRDE2 during seed germination. In summary, our results suggest that FtRDE2 can serve as a promising candidate for the molecular breeding of a TB variety with minimal bitterness.

Carbon sinks/sources' spatiotemporal heterogeneity and their spatial response to economic-social factors in relatively developed regions: A case of China's coastal area.

With the global climate change, carbon reduction in economically active regions has gradually become a focus of attention and its underlying drivers were essential for understanding alterations in ecosystems in response to human behavior. However, the exploration of Carbon Sinks/Sources Patterns (CSSP) in an Economic-Social context was lacking. Distinguished from traditional Net Ecosystem Productivity (NEP) estimation methods, we optimized model parameters, adjusted estimation logic, and revealed CSSP more reasonably. Moreover, spatial econometric model was used to reveal the spatial effects mechanism of Economic-Social Development on CSSP. Over the past 20 years, we revealed that: (a) The pattern of NEP exhibited distinct spatial heterogeneity, with higher sinks observed in the north and offshore regions. It demonstrated regular cyclic fluctuations, averaging a 3-4-year cycle, featuring a gradual ascent followed by a rapid descent; (b) The Carbon Sequestration Capacity (CSC) of vegetation significantly increased. Based on the carbon sink properties, the study area was distinctly divided into three clusters; (c) CSSP have been profoundly affected by economic-social factors. Economic growth and industrial structure optimization contributed to the enhancement of CSC, but population aggregation and urban expansion had negative impacts. The direct effect of innovation capacity and the spatial spillover effect of industrial structure optimization were negative. Overall, exploring CSSP against the backdrop of economic-social factors not only provides a new perspective for understanding the regularities of change and the underlying mechanisms driven by human factors but also offers valuable insights for achieving sustainable development and green growth in other coastal regions globally.

Novel Digital SERS-Microfluidic Chip for Rapid and Accurate Quantification of Microorganisms.

In this work, we present a simple and novel digital surface-enhanced Raman spectroscopy (SERS)-microfluidic chip designed for the rapid and accurate quantitative detection of microorganisms. The chip employs a high-density inverted pyramid microcavity (IPM) array to separate and isolate microbial samples. The presence or absence of target microorganisms is determined by scanning the IPM array using SERS and identifying the characteristic Raman bands. This approach allows for the "digitization" of the SERS response of each IPM, enabling quantification through the application of mathematical statistical techniques. Significantly, precise quantitative detection of yeast was achieved within a concentration range of 106-109 cells/mL, with the maximum relative standard deviation from the concentration calibrated by the cultivation method being 5.6%. This innovative approach efficiently addresses the issue of irregularities in SERS quantitative detection, which arises due to fluctuations in SERS intensity and poor reproducibility. We strongly believe that this digital SERS-microfluidic chip holds immense potential for diverse applications in the rapid detection of various microorganisms, including pathogenic bacteria and viruses.

Single-molecule magnet behavior in heterometallic decanuclear [Ln2Fe8] (Ln = Y, Dy, Ho, Tb, Gd) coordination clusters.

Five decanuclear lanthanide-iron clusters, formulated as [Ln2Fe8(hmp)10(µ2-OH)4(µ3-OH)2(µ4-O)4(H2O)6]·6ClO4·xH2O (x ≈ 8, Ln = Y for 1; x ≈ 6, Ln = Dy for 2; x ≈ 6, Ln = Ho for 3; x ≈ 7, Ln = Tb for 4; x ≈ 7, Ln = Gd for 5, Hhmp = 2-(hydroxymethyl)pyridine), have been synthesized and structurally characterized. Single-crystal structural analysis reveals that the cluster consists of six face-sharing defective cubane units. Dynamic magnetic investigations indicated that cluster 2 exhibits single-molecule magnet behavior under a zero dc field eliciting an effective energy barrier of Ueff = 17.76 K and a pre-exponential factor of τ0 = 7.93 × 10-8 s. Investigation of the performance of a series of FeIII-DyIII SMMs indicates that the relatively low energy barrier in 2 is associated with the weak ferromagnetic coupling between FeIII and DyIII ions, while the strength of ferromagnetic interaction in these clusters is mainly related to the bond distances between DyIII and O atoms coordinated to FeIII ions. Clusters 3 and 4 exhibit similar dual relaxation pathways under their respective optimal external applied dc field, where the direct relaxation process occurs in the low-frequency area, which impedes the extraction of the Ueff, while the secondary relaxation process appears at a higher frequency, which is probably a connection with intermolecularly driven relaxation. Our findings offer a magneto-structural correlation model for further investigating the single-molecule magnet behavior in lanthanide-iron systems.

Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y).

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

Comparison of errors in ablation depth calculation after myopic femtosecond laser in situ keratomileusis in patients with different degrees of myopia: a prospective study.

BACKGROUND: To investigate the difference between the predicted preoperative corneal ablation depth and the measured ablation depth for femtosecond laser in situ keratomileusis (FS-LASIK) in patients with different degrees of myopia, and to analyze the source of the difference. METHODS: A total of 55 patients (109 eyes) were included in this study. Multiple logistics regression was applied to analyze the sources affecting postoperative refractive outcomes. The difference between the preoperative predicted corneal ablation depth and the 1-day postoperative ablation depth in patients with different degrees of myopia was explored using linear regression. Corneal biomechanical parameters influencing error in ablation depth calculation were examined using multiple linear regression. RESULTS: One hundred and nine eyes were divided into low to moderate myopia (55 eyes, myopia of 6 D or less), high myopia (45 eyes, myopia ranging from 6 D to a maximum of 9 D), and very high myopia group (9 eyes, myopia greater than 9 D) based on preoperative refractive error (spherical equivalent). Postoperative visual outcomes were comparable among the three groups of patients, with no significant difference in uncorrected visual acuity (UCVA). We did find notable disparities in spherical equivalent (SE) and central corneal thickness (CCT) in patients with different degrees of myopia at 1 day postoperatively (all p < 0.001). Logistic regression analysis showed that error in ablation depth calculation was an independent risk factor for refractive outcomes one day after surgery (OR = 1.689, 95% CI: 1.366 - 2.089). There was a substantial discrepancy in error in ablation depth calculation at 1 day postoperatively between the three groups. The measured ablation depth of the laser platform was lower than the predicted ablation depth in the low to moderate myopia and very high myopia groups, but the opposite was true in the high myopia group. Pre-operative SE (p < 0.001) and corneal front minimum radius of curvature (Front Rmin) (p = 0.007) obviously influenced the error in ablation depth calculation. CONCLUSIONS: Error in ablation depth calculation values vary significantly between patients with different degrees of myopia and correlate highly with preoperative SE and Front Rmin. At the same time, the available evidence suggests that error in ablation depth calculation is an influential factor in postoperative refractive status, so it is imperative to control error in ablation depth calculation.

Evaluating Tartary Buckwheat Genotypes with High Callus Induction Rates and the Transcriptomic Profiling during Callus Formation.

Due to their complex genotypes, low in vitro regeneration rates, and difficulty in obtaining transgenic plants, studies concerning basic biological research and molecular breeding in Tartary buckwheat (TB) are greatly limited. In this study, the hypocotyls of 60 genotypes of TB (TBC1~60) were used as explants. Of these, TBC14 was selected due to a high callus induction rate of 97.78% under dark and a proliferation coefficient (PC) of 28.2 when cultured on MS medium supplemented with 2.0 mg/L of 2,4-D and 1.5 mg/L of 6-BA. Subsequently, the samples of the calli obtained from TBC14 were collected at 0, 10, 20, and 30 d, and their transcriptomes were sequenced where identified. GO enrichment led to the detection of the most significant active gene set, which was the DNA binding transcription factor activity. The DEGs related to the pathways concerning metabolism, the biosynthesis of secondary metabolites, and hormone signal transduction were the most enriched in the KEGG database. The sets of MYB, AP2/ERF, and bHLH TFs exhibited the highest number of DEGs. Using this enrichment analysis, 421 genes encoding TFs, 47 auxin- and cytokinin-related genes, and 6 signal transduction-associated genes were screened that may play significant roles in callus formation (CF) in TB. Furthermore, FtPinG0008123200.01 (bZIP), a key gene promoting CF, was screened in terms of the weighted gene co-expression network associated with the various stages of CF. Our study not only provides valuable information about the molecular mechanism of CF but also reveals new genes involved in this process.