A smartphone-adaptable fluorescent probe for visual monitoring of fish freshness and its application in fluorescent dyes.

Due to increasing food safety issues, developing intelligent, on-site, and visual methods for detecting fish freshness has attracted significant attention. Here, we have prepared a benzo[h]chromene derivative BCN that can visually detect 12 biogenic amines (BAs) with high sensitivity. The mechanism for recognizing cadaverine (Cad) is that the probe reacts with Cad to produce a Schiff base derivative, which alters the charge distribution within the molecule, resulting in significant colorimetric and fluorescence changes. The sensing label BCN/FPS was prepared by loading the probe BCN on filter paper, and a visual detection platform was constructed by combining it with a smartphone. By monitoring the correspondence between label color and TVB-N content, a working curve of (R + B)/(R + B + G) with TVB-N content was obtained, enabling visual evaluation of salmon freshness using only a mobile phone. In addition, based on the good solubility and processability of BCN, its application in fluorescent dyes including impregnating dyes, printing inks, coatings, and flexible films has been explored, which opens up new directions for the application of BCN. Therefore, BCN has the potential for real-time monitoring of meat freshness and preparation of fluorescent materials.

Study on trade-offs and synergies of rural ecosystem services in the Tacheng-Emin Basin, Xinjiang, China: Implications for zoning management of rural ecological functions.

Rural areas are the main source of ecosystem services in arid and semi-arid areas, and ecosystem services are the background conditions for rural revitalization. In this study, the spatial pattern of key ecosystem services in the countryside was assessed, and the trade-offs and synergistic relationships among ecosystem services were investigated, using the Tacheng-Emin Basin in China as the study area. Finally, the types of ecological function zoning and development strategies for the countryside are proposed. The results showed that: (1) the area of ecological land was large, and the average land use intensity was 2.48, which belonged to the medium intensity. (2) The mean values of the six ecosystem services are all in the middle and lower classes, and the spatial distribution of the five ecosystem services is similar, except for food production. (3) Except for grain production, the other five ecosystem services showed positive feedback to elevation. The other five ecosystem services are synergistic, and there are trade-offs between grain production and other ecosystem services. In the nonlinear interaction mechanism of ecosystem services, the fluctuation constraint occupies the largest proportion. (4) At smaller spatial scales, there are more types of ecosystem service clusters. Combining the results of the study, the villages in the study area can be categorized into five types. This study formulates five priority levels of rural ecological revitalization and proposes different development recommendations for the sustainable development of each type of village. This study is helpful for the fine management of land resources and the revitalization of rural ecology and provides a reference for the sustainable development of ecosystem services in arid and semi-arid areas.

Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

Corrigendum to 'Characterization of lipid-based nanomedicines at the single-particle level' [Fundamental Research 3 (2023) 488-504].

[This corrects the article DOI: 10.1016/j.fmre.2022.09.011.].

Construction and Validation of Chicken Immune scFv Antibody Library against Helicobacter pylori.

Accurate diagnostic techniques and effective therapeutic methods are required to treat H. pylori. The application of chicken single-chain variable fragment (scFv) antibodies may diagnose and treat H. pylori. This study used the phage display technique to construct a chicken-derived immune scFv antibody library against H. pylori. Total RNA was extracted from the spleens of five immunized chickens and reverse transcribed into cDNA. A fragment of scFv was produced by overlap extension PCR and cloned into a pHEN2 phagemid vector. After the package with the M13KO7 helper phage, the recombinant HpaA protein was used as a target antigen to validate the screening ability of our antibody library by bio-panning. The dilution counting results showed that the size of the primary antibody library was estimated to be 1 × 109 cfu/mL. PCR analysis of 47 clones from the library revealed that about 100% of the clones were positive with scFv fragments, and there were no identical sequences, indicating the good diversity of the antibody library. After three rounds of bio-panning, high-affinity antibodies against recombinant HpaA protein were successfully obtained. The selected antibody specifically recognized HpaA protein in nine different H. pylori strains, confirming the screening ability of our library. The chicken immune scFv antibody library against H. pylori was successfully constructed, and the antibody library's screening ability was validated by selecting specific scFv antibodies against recombinant HpaA and clinical strains. It provided a simple and rapid method to obtain antibodies against H. pylori for diagnosis or treatment.

Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy.

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Δ Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

A novel fluorescence probe for ultrafast detection of SO2 derivatives/biogenic amines and its multi-application: Detecting food and fish freshness, fluorescent dye and bioimaging.

In this study, we have effectively prepared a novel fluorescent probe named HDXM based on benzopyran derivatives for the ultrafast detection (within 3 s) of SO2 derivatives or biogenic amines. HDXM showed a noticeable color change after the addition of SO2 derivatives (from purple to colorless) or biogenic amines (from purple to blue), indicating that HDXM can identify two analytes with the naked eye. It is worth noting that HDXM can be used to detect SO2 derivatives in actual sugar samples, and to image HSO3-/SO32- in living cells. More importantly, sensing labels (HDXM-loaded filter paper or agarose hydrogel) enable real-time visual monitoring of salmon freshness through colorimetric and fluorescence dual channels. Compared with the Chinese national standard method, the sensing label is an effective tool for evaluating the freshness of fish. Benefiting from its excellent solubility and fluorescence performance, HDXM can be used as a versatile fluorescent material in various applications, including flexible films, glass coatings, impregnating dyes, printing, and fingerprint ink. HDXM is expected to be a promising and valuable multifunctional tool for food safety and fluorescent materials.

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry.

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

Genomic diversity and taxonomic marker for Arcobacter species.

Arcobacter was recognized as an emerging enteropathogen and controversies regarding its classification persisted. This study aimed to reevaluate the taxonomy of Arcobacter utilizing the 16S rRNA gene, 23S rRNA gene, single-copy orthologous genes, as well as genomic indices such as Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (isDDH). The taxonomy of this genus was reevaluated in this study using multiple indices with a dataset of 371 genomes comprising 34 known species and 14 potentially new species. Good discrimination could be achieved only in some species but not for the species with higher sequence similarity using the comparisons of the 16S rRNA gene and 23S rRNA gene sequences. A high-accuracy phylogenomic approach for Arcobacter was established using 84 single-copy orthologous genes obtained through various bioinformatics methods. One marker gene (gene711), which was found to possess the same distinguishing ability as ANI, isDDH, and single-copy orthologous methods, was identified as a reliable locus for inferring the phylogeny of the genus. The effective species classification was achieved by employing gene711 with a sequence similarity exceeding 96%, even for species like A. cloacae, A. lanthieri, and A. skirrowii, which exhibited ambiguous classification using ANI and isDDH. Additionally, excellent subspecies categorizing among A. cryaerophilus could be distinguished using gene711. In conclusion, this framework strategy had the potential advantage of developing rapid species identification, particularly for highly variable species, providing a novel insight into the behavior and characteristics of Arcobacter.

Rapid, Selective Extraction of Silver from Complex Water Matrices with a Metal-Organic Framework/Oligomer Composite Constructed via Supercritical CO2.

Every year vast quantities of silver are lost in various waste streams; this, combined with its limited, diminishing supply and rising demand, makes silver recovery of increasing importance. Thus, herein, we report a controllable, green process to produce a host of highly porous metal-organic framework (MOF)/oligomer composites using supercritical carbon dioxide (ScCO2 ) as a medium. One resulting composite, referred to as MIL-127/Poly-o-phenylenediamine (PoPD), has an excellent Ag+ adsorption capacity, removal efficiency (>99 %) and provides rapid Ag+ extraction in as little as 5 min from complex liquid matrices. Notably, the composite can also reduce sliver concentrations below the levels (<0.1 ppm) established by the United States Environmental Protection Agency. Using theoretical simulations, we find that there are spatially ordered polymeric units inside the MOF that promote the complexation of Ag+ over other common competing ions. Moreover, the oligomer is able to reduce silver to its metallic state, also providing antibacterial properties.

Quantitative assessment of lipophilic membrane dye-based labelling of extracellular vesicles by nano-flow cytometry.

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.

A red fluorescence probe for reversible detection of HSO3-/H2O2 and its application in food samples and bioimaging.

Reducing agent SO2 and oxidant H2O2 are two essential substances in cells, and the balance between them is closely related to the survival of cells. SO2 derivative HSO3- is often used as food additive. Therefore, simultaneous detection of SO2 and H2O2 is of great significance in biology and food safety. In this work, we successfully developed a mitochondria-targeted red fluorescent probe (HBTI), which has excellent selectivity, high sensitivity and large Stokes shift (202 nm). HBTI and HSO3-/SO32- undergo Michael addition on the unsaturated C=C bond, and the addition product (HBTI-HSO3-) can react with H2O2 to restore the conjugated structure. Fluorescence changes from red to non-emissive and then restores to red, and can be detected quickly and visually. In addition, HBTI has been successfully targeted mitochondria, and achieved dynamic reversible response to SO2/H2O2 in living cells, and has been successfully applied to detect SO2 in food samples.

A food poisoning caused by ST7 Staphylococcal aureus harboring sea gene in Hainan province, China.

ST7 Staphylococcus aureus is highly prevalent in humans, pigs, as well as food in China; however, staphylococcal food poisoning (SFP) caused by this ST type has rarely been reported. On May 13, 2017, an SFP outbreak caused by ST7 S. aureus strains occurred in two campuses of a kindergarten in Hainan Province, China. We investigated the genomic characteristics and phylogenetic analysis of ST7 SFP strains combined with the 91 ST7 food-borne strains from 12 provinces in China by performing whole-genome sequencing (WGS). There was clear phylogenetic clustering of seven SFP isolates. Six antibiotic genes including blaZ, ANT (4')-Ib, tetK, lnuA, norA, and lmrS were present in all SFP strains and also showed a higher prevalence rate in 91 food-borne strains. A multiple resistance plasmid pDC53285 was present in SFP strain DC53285. Among 27 enterotoxin genes, only sea and selx were found in all SFP strains. A ФSa3int prophage containing type A immune evasion cluster (sea, scn, sak, and chp) was identified in SFP strain. In conclusion, we concluded that this SFP event was caused by the contamination of cakes with ST7 S. aureus. This study indicated the potential risk of new emergencing ST7 clone for SFP.

Herbaspirillum sp. ZXN111 Colonization Characters to Different Tea Cultivars and the Effects on Tea Metabolites Profiling on Zijuan (Camellia sinensis var. assamica).

Herbaspirillum sp. ZXN111 and its mutants (Δacc, Δtyrb, and Δacc-tyrb), which show PGP activity on Zijuan, were tested for tea plants' colonization characteristics and the strain-dependent response of tea metabolites. The results showed that strain ZXN111 could widely colonize in different tea cultivars of Zijuan, Yunkang-10, Longjin 43, and Shuchazao, but with significant colonization preference to Zijuan, which might be ascribed to anthocyanins' chemotaxis. After 9 weeks of co-cultivation, l-theanine and theobromine in Zijuan leaves that were inoculated with wild-type ZXN111 were decreased, while theobromine, caffeine, and l-theanine that were inoculated with mutant Δacc were increased; especially l-theanine increased much significantly. Metabolomics analysis showed that tea metabolite profiling of inoculant groups was clearly separated from the control; therein, the flavanols were downregulated in ZXN111 and Δacc groups, but the l-theanine of the Δacc group was significantly upregulated compared to control and ZXN111 groups. These results indicated that strain ZXN111, especially of mutant Δacc, improved Zijuan tea flavor.

Linezolid-Resistant Enterococcus faecalis of Chicken Origin Harbored Chromosome-Borne optrA and Plasmid-Borne cfr, cfr(D), and poxtA2 Genes.

The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an Enterococcus faecalis isolate QZ076, cocarrying optrA, cfr, cfr(D), and poxtA2 genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using E. faecalis JH2-2 and clinical methicillin-resistant Staphylococcus aureus (MRSA) 109 as recipients. E. faecalis QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with optrA located in the chromosomal DNA. The gene cfr was located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes cfr(D) and poxtA2 were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The cfr-carrying plasmid pQZ076-1 could transfer from E. faecalis QZ076 to E. faecalis JH2-2, along with the cfr(D)- and poxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA, cfr, cfr(D), and poxtA2] being simultaneously present in the same E. faecalis isolate. The location of the cfr gene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the cfr-carrying pheromone-responsive conjugative plasmid in E. faecalis was also able to mobilize the interspecies transfer of the cfr(D)- and poxtA2-cocarrying plasmid between enterococci and staphylococci. IMPORTANCE In this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA, cfr, cfr(D), and poxtA2] was identified in an E. faecalis isolate of chicken origin. The association of the cfr gene with a novel pseudocompound transposon Tn7515 integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genes cfr(D) and poxtA2 on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such as cfr, cfr(D), and poxtA2, among Gram-positive pathogens.

Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles.

Correlated analysis of multiple biochemical parameters at the single-particle level and in a high-throughput manner is essential for insights into the diversity and functions of biological nanoparticles (BNPs), such as bacteria and subcellular organelles. To meet this challenge, we developed a highly sensitive spectral nano-flow cytometer (S-nFCM) by integrating a spectral recording module to a laboratory-built nFCM that is 4-6 orders of magnitude more sensitive in side scattering detection and 1-2 orders of magnitude more sensitive in fluorescence detection than conventional flow cytometers. An electron-multiplying charge-coupled device (EMCCD) was used to acquire the full fluorescence spectra of single BNPs upon holographic grating dispersion. Up to 10,000 spectra can be collected in 1 min with 2.1 nm resolution. The precision, linearity, and sensitivity were examined. Complete discernment of single influenza viruses against the background signal, discrimination of different strains of marine cyanobacteria in a mixed sample based on their spectral properties of natural fluorescence, classification of bacterial categories exhibiting different patterns of antigen expression, and multiparameter analysis of single mitochondria for drug discovery were successfully demonstrated.

A compendium of single extracellular vesicle flow cytometry.

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

CsGDH2.1 negatively regulates theanine accumulation in late-spring tea plants (Camellia sinensis var. sinensis).

Theanine, a unique and the most abundant non-proteinogenic amino acid in tea plants, endows tea infusion with the umami taste and anti-stress effects. Its content in tea correlates highly with green tea quality. Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring. However, how the decrease is regulated is largely unknown. In a genetic screening, we observed that a yeast mutant, glutamate dehydrolase 2 (gdh2), was hypersensitive to 40 mM theanine and accumulated more theanine. This result implied a role of CsGDH2s in theanine accumulation in tea plants. Therefore, we identified the two homologs of GDH2, CsGDH2.1 and CsGDH2.2, in tea plants. Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant. Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues. Importantly, CsGDH2.1 expression was low in early spring, and increased significantly in late spring, in the new shoots of tea plants. These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots. Moreover, the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity, and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants. These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring, and provides a functional gene for improving late-spring green tea quality.