Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

Multiomics integrated with sensory evaluations to identify characteristic aromas and key genes in a novel brown navel orange (Citrus sinensis).

'Zong Cheng' navel orange (ZC) is a brown mutant of Lane Late navel orange (LL) and emits a more pleasant odor than that of LL. However, the key volatile compound of this aroma and underlying mechanism remains unclear. In this study, sensory evaluations and volatile profiling were performed throughout fruit development to identify significant differences in sensory perception and metabolites between LL and ZC. It revealed that the sesquiterpene content varied significantly between ZC and LL. Based on aroma extract dilution and gas chromatography-olfactometry analyses, the volatile compound leading to the background aroma of LL and ZC is d-limonene, the orange note in LL was mainly attributed to octanal, whilst valencene, ß-myrcene, and (E)-ß-ocimene presented balsamic, sweet, and herb notes in ZC. Furthermore, Cs5g12900 and six potential transcription factors were identified as responsible for valencene accumulation in ZC, which is important for enhancing the aroma of ZC.

Flavor characterization of Citri Reticulatae Pericarpium (Citrus reticulata 'Chachiensis') with different aging years via sensory and metabolomic approaches.

Guangchenpi (GCP), which is the peel of Citrus reticulata 'Chachiensis', is widely used as an herbal medicine, tea and food ingredient in southeast Asia. Prolonging its aging process results in a more pleasant flavor and increases its profitability. Through the integration of sensory evaluation with flavoromic analysis approaches, we evaluated the correlation between the flavor attributes and the profiles of the volatiles and flavonoids of GCP with various aging years. Notably, d-limonene, γ-terpinene, dimethyl anthranilate and α-phellandrene were the characteristic aroma compounds of GCP. Besides, α-phellandrene and nonanal were decisive for consumers' perception of GCP aging time due to changes of their odor activity values (OAVs). The flavor attributes of GCP tea liquid enhanced with the extension of aging time, and limonene-1,2-diol was identified as an important flavor enhancer. Combined with machine learning models, key flavor-related metabolites could be developed as efficient biomarkers for aging years to prevent GCP adulteration.

Screening Universal Stress-Response Terpenoids and Their Biosynthetic Genes via Volatile and Transcriptomic Profiling in Citrus.

Volatile terpenoids accumulate in citrus and play important roles in plant defense against various stressors. However, the broad-spectrum response of terpenoid biosynthesis to ubiquitous stressors in citrus has not been comparatively investigated. In this study, volatile terpenoids were profiled under six stressors: high temperature, citrus miner, citrus red mite, citrus canker, Alternaria brown spot, and huanglongbing (HLB). Significant content changes in 15 terpenoids, including ß-ocimene, were observed in more than four of the six stressors, implying their possibly universal stress-response effects. Notably, the emission of terpenoids, including ß-caryophyllene, ß-ocimene, and nerolidol glucoside, was significantly increased by HLB in HLB-tolerant "Shatian" pomelo leaves. The upregulation of CgTPS1 and CgTPS2 and their characterization in vivo identified them as mono- or sesquiterpenoid biosynthetic genes. This study provides a foundation for determining stress resistance mechanisms in citrus and biopesticide designations for future industrial applications.

Exploring the Application and Prospects of Synthetic Biology in Engineered Living Materials.

At the intersection of synthetic biology and materials science, engineered living materials (ELMs) exhibit unprecedented potential. Possessing unique "living" attributes, ELMs represent a significant paradigm shift in material design, showcasing self-organization, self-repair, adaptability, and evolvability, surpassing conventional synthetic materials. This review focuses on reviewing the applications of ELMs derived from bacteria, fungi, and plants in environmental remediation, eco-friendly architecture, and sustainable energy. The review provides a comprehensive overview of the latest research progress and emerging design strategies for ELMs in various application fields from the perspectives of synthetic biology and materials science. In addition, the review provides valuable references for the design of novel ELMs, extending the potential applications of future ELMs. The investigation into the synergistic application possibilities amongst different species of ELMs offers beneficial reference information for researchers and practitioners in this field. Finally, future trends and development challenges of synthetic biology for ELMs in the coming years are discussed in detail.

Chlorophyll maxima layer in a large subtropical reservoir (Xinanjiang Reservoir): Spatial development process and limitation by CO2 and phosphorus.

In marine investigations, the maximum chlorophyll-a (Chla) concentration is often reported to occur at a specific depth below the ocean surface, a phenomenon known as subsurface Chla maxima (SCM). However, SCM has long been overlooked in artificial reservoirs, which may lead to a serious underestimation of the primary productivity level and trophic status of reservoirs. To better understand the temporal and spatial variability of SCM and the mechanisms leading to SCM development, this study conducted a detailed survey in a large subtropical reservoir (Xinanjiang Reservoir, XAJR) from September 2020 to August 2021. The seasonal thermal stratification, in situ variables (WT, pH, DO and Chla), nutrient concentrations (DSi, NO3-, DIP and DCO2), Chla maxima depth and magnitude of the riverine region (S1), transition region (S2) and the central part of the XAJR (S3 and S4) were all thoroughly investigated. Thermal stratification and SCM in XAJR exhibited significant seasonal and spatial heterogeneity. Phytoplankton biomass in the epilimnion was limited by dissolved CO2 from June to October in the warm seasons, while it was primarily limited by phosphorus in the other seasons, according to the nutrient limitation analysis. Along the water column, dissolved CO2 limitation occurred mainly above the SCM layer, and the water column below the SCM layer gradually transitioned from dissolved CO2 limitation to phosphorus limitation. Furthermore, as the thermal stratification developed, the upstream water mass moves along the middle of the water column as density flow toward the reservoir, providing nutrients for the development of the SCM. This research contributes to a better understanding of the temporal and spatial variation of SCM and nutrient supply in deep and large stratified reservoirs.

Study on carbon dioxide emission from reservoirs with different regulation types and its empirical prediction model.

The construction of artificial reservoirs with various regulation types on river is currently an important form of comprehensive utilization of water energy and water resources in river basins. The type of regulation is important in controlling the residence time, which in turn affects the photosynthesis-respiration balance in the water. This process has a significant impact on carbon dioxide (CO2) emissions from reservoirs. In this study, seasonal observations were carried out from September 2020 to July 2021 at five artificial reservoirs in the Qiantang River Basin, eastern China, to reveal the characteristics of CO2 emission from the water-air interface of reservoirs with different regulating types. The results showed that the annual average CO2 emission flux of the studied reservoirs varied significantly, ranging from 4.2 to 155.3 mmol m-2 day-1 with an average of 48.4 mmol m-2 day-1, which also had a significant negative correlation with the hydraulic retention time. While downstream of the dam, the annual average CO2 emission flux was quite high with a range of 105.8 to 543.0 mmol m-2 day-1, averaging 381.6 mmol m-2 day-1. This is mainly due to the release of water with high-concentration CO2 from the bottom of the reservoir. Additionally, using related data of reservoirs around the world, a CO2 emission model with hydraulic retention time, air temperature, and reservoir age as the primary parameters was developed, which was conducive to evaluate reservoir CO2 emissions on a larger scale and provided theoretical support for effective reservoir management.

Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system.

OBJECTIVE: To generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis. METHODS: CRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis. RESULTS: Six knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis. CONCLUSION: Large fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.

National survey on internal quality control for tumour markers in clinical laboratories in China.

INTRODUCTION: This survey was initiated to obtain knowledge on the current situation of internal quality control (IQC) practice for tumour markers (TMs) in China. Additionally, we tried to acquire the most appropriate quality specifications. MATERIALS AND METHODS: This survey was a current status survey. The IQC information had been collected via online questionnaires. All of 1821 clinical laboratories which participated in the 2016 TMs external quality assessment (EQA) programme had been enrolled. The imprecision evaluation criteria were the minimal, desirable, and optimal allowable imprecisions based on biological variations, and 1/3 total allowable error (TEa) and 1/4 TEa. RESULTS: A total of 1628 laboratories answered the questionnaires (89%). The coefficients of variation (CVs) of the IQC of participant laboratories varied greatly from 1% (5th percentile) to 13% (95th percentile). More than 82% (82 - 91%) of participant laboratories two types of CVs met 1/3 TEa except for CA 19-9. The percentiles of current CVs were smaller than cumulative CVs. A number of 1240 laboratories (76%) reported their principles and systems used. The electrochemiluminescence was the most used principle (45%) and had the smallest CVs. CONCLUSIONS: The performance of laboratories for TMs IQC has yet to be improved. On the basis of the obtained results, 1/3 TEa would be realistic and attainable quality specification for TMs IQC for clinical laboratories in China.