Dissection of the spatial dynamics of biosynthesis, transport, and turnover of major amino acids in tea plants (Camellia sinensis).

High levels of free amino acids (AAs) in tea leaves are crucial for tea flavor and health function; however, the dynamic AA biosynthesis, transport, and turnover in tea plants remain elusive. Here we dissected whole tea plants for these dynamics by assessing AA profiles and transcriptomes of metabolic pathway genes in tea roots, stems, and leaves and revealing their distinctive features with regard to AA synthesis, transport, and degradation/recycling. Nitrogen assimilation dominated in the roots wherein glutamine (Gln), theanine, and arginine (Arg) were actively synthesized. Arg was transported into trunk roots and stems, together with Glu, Gln, and theanine as the major AAs in the xylem sap for long-distance root-to-leaf transport. Transcriptome analysis revealed that genes involved in Arg synthesis were highly expressed in roots, but those for Arg transport and degradation were highly expressed in stems and young leaves, respectively. CsGSIa transcripts were found in root meristem cells, root, stem and leaf vascular tissues, and leaf mesophyll where it appeared to participate in AA synthesis, transport, and recycling. Overexpression of CsGSIa in tea transgenic hairy roots and knockdown of CsGSIa in transgenic hairy roots and tea leaves produced higher and lower Gln and theanine than wild-type roots and leaves, respectively. This study provides comprehensive and new insights into AA metabolism and transport in the whole tea plant.

Hydrolysis of Beef Sarcoplasmic Protein by Dry-Aged Beef-Isolated Penicillium oxalicum and Its Associated Metabolic Pathways.

Yanbian cattle have a unique meat flavor, and high-grade meat is in short supply. Therefore, in this study, we aimed to improve the added value of Yanbian cattle low-fat meat and provide a theoretical reference for the subsequent development of an excellent starter. Rump meat from Yanbian cattle was dry-aged and then screened for protease-producing fungi. Three protease-producing fungi (Yarrowia hollandica (D4 and D11), Penicillium oxalicum (D5), and Meesziomyces ophidis (D20)) were isolated from 40 d dry-aged beef samples, and their ability to hydrolyze proteins was determined using bovine sarcoplasmic protein extract. SDS-PAGE showed that the ability of Penicillium oxalicum (D5) to degrade proteins was stronger than the other two fungi. In addition, the volatile component content of sarcoplasmic proteins in the D5 group was the highest (45.47%) and comprised the most species (26 types). Metabolic pathway analysis of the fermentation broth showed that phenylalanine, tyrosine, and tryptophan biosynthesis was the most closely related metabolic pathway in sarcoplasmic protein fermentation by Penicillium oxalicum (D5). Dry-aged beef-isolated Penicillium oxalicum serves as a potential starter culture for the fermentation of meat products.

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology.

Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels, including small RNAs (sRNAs), riboswitches, and antisense RNAs. Notably, the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription, post-transcription or translation processes. Leveraging and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic biology. While previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica, this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria, notably Bacillus subtilis. Furthermore, we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology, employing B. subtilis as a foundational chassis.

What Fosters School Connectedness? The Roles of Classroom Interactions and Parental Support.

Although research has identified the impact of school connectedness on a variety of outcomes for adolescents, much less work has focused on identifying its precursors. This study examined the relative influences of classroom interactions and parental support on elements of school connectedness among a sample of 4838 students (Mage = 15.84, SD = 0.29; 49.1% female) in the United States from the Programme for International Student Assessment (PISA) 2018 data. The results showed that three domains of classroom interactions (i.e., classroom management, instructional support, and emotional support) and parental support played unique roles in predicting school connectedness (i.e., teacher support and school belonging). Specifically, classroom management positively predicted both teacher support and school belonging; instructional support, especially directed instruction, positively predicted teacher support; emotional support was unrelated to teacher support and school belonging. Parental support positively predicted school belonging, but not teacher support. Overall, these findings highlight the roles of both teachers and parents in providing developmentally appropriate support to facilitate school connectedness.

The relation of bilingual cognitive skills to the second language writing performance of primary grade students.

The purposes of the current study were to examine the effects of cognitive and reading skills (i.e., working memory [WM], oral language development [OLD], and reading skills) on second language (L2) writing performance as well as the changes in these relationships across different grades among Spanish-speaking children learning English. A battery of measures assessing English and Spanish WM, OLD, reading skills, and English writing were administered to 494 English learners in Grades 1 to 3. Path analysis was conducted for each grade separately in both English and Spanish models. The findings indicated that the relationships between English writing performance and English cognitive and reading skills became stronger as the grades increased. However, the relationships between English writing and the Spanish cognitive and reading determinants were mixed, indicating a statistically significant relationship with Spanish WM and reading skills for Grade 2 and 3 students but not with OLD across all grades. Implications for L2 writing development are discussed.

Association of seizures with COVID-19 infection in underage during the pandemic: A systematic review and meta-analysis.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is a contagious infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused worldwide transmission. The aim of this systematic review and meta-analysis was to investigate the morbidity and characteristics of seizures after SARS-CoV-2 infection in underage (≤18 y) and to provide valuable reference material for subsequent clinical treatment. METHODS: PubMed/MEDLINE, Cochrane, and EMBASE databases were searched up to 10th May 2023. We utilized the search strategy of medical subject headings combined with entry terms to search all related literatures. RESULTS: The meta-analysis was performed according to PRISMA reporting guidelines. Risk of bias was assessed using the Newcastle-Ottawa Scale (NOS) and the Agency for Healthcare Research and Quality (AHRQ). A total of 12 articles were selected, including 4153 subjects and 333 seizure-prone minor patients with COVID-19. The morbidity of seizures after SARS-CoV-2 infection in immature patients was approximately 8.2 (95 % CI, 4.7%-12.4 %). By subgroup analysis, we know that the morbidity of male, Americas, with fever and first occurrence of seizures were 4.2% (95 % CI, 0.4-10.5 %), 4.6 % (95 % CI, 0.4 %-11.7 %), 5.4 % (95 % CI, 2.6 %-9.1 %) and 3.7 % (95 % CI, 0.7 %-8.2 %), respectively. Generalized seizures are the main type of seizures (80.6 %). CONCLUSIONS: Seizures can be caused by SARS-CoV-2 infection in underage groups, with a pooled morbidity of 8.2% and a higher morbidity in females, in African regions, in febrile groups and during 2022-2023. In addition, generalized seizures being the predominant seizure type.

CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis).

Anthocyanin and catechin production in tea (Camellia sinensis) leaves can positively affect tea quality; however, their regulatory mechanisms are not fully understood. Here we report that, while the CsMYB75- or CsMYB86-directed MYB-bHLH-WD40 (MBW) complexes differentially activate anthocyanin or catechin biosynthesis in tea leaves, respectively, CsMYBL2a and CsMYBL2b homologs negatively modified the light- and temperature-induced anthocyanin and catechin production in both Arabidopsis and tea plants. The MBW complexes activated both anthocyanin synthesis genes and the downstream repressor genes CsMYBL2a and CsMYBL2b. Overexpression of CsMYBL2b, but not CsMYBL2a, repressed Arabidopsis leaf anthocyanin accumulation and seed coat proanthocyanin production. CsMYBL2b strongly and CsMYBL2a weakly repressed the activating effects of CsMYB75/CsMYB86 on CsDFR and CsANS, due to their different EAR and TLLLFR domains and interactions with CsTT8/CsGL3, interfering with the functions of activating MBW complexes. CsMYBL2b and CsMYBL2a in tea leaves play different roles in fine-tuning CsMYB75/CsMYB86-MBW activation of biosynthesis of anthocyanins and catechins, respectively. The CsbZIP1-CsmiR858a-CsMYBL2 module mediated the UV-B- or cold-activated CsMYB75/CsMYB86 regulation of anthocyanin/catechin biosynthesis by repressing CsMYBL2a and CsMYBL2b. Similarly, the CsCOP1-CsbZIP1-CsPIF3 module, and BR signaling as well, mediated the high temperature repression of anthocyanin and catechin biosynthesis through differentially upregulating CsMYBL2b and CsMYBL2a, respectively. The present study provides new insights into the complex regulatory networks in environmental stress-modified flavonoid production in tea plant leaves.

Aluminum and Fluoride Stresses Altered Organic Acid and Secondary Metabolism in Tea (Camellia sinensis) Plants: Influences on Plant Tolerance, Tea Quality and Safety.

Tea plants have adapted to grow in tropical acidic soils containing high concentrations of aluminum (Al) and fluoride (F) (as Al/F hyperaccumulators) and use secret organic acids (OAs) to acidify the rhizosphere for acquiring phosphorous and element nutrients. The self-enhanced rhizosphere acidification under Al/F stress and acid rain also render tea plants prone to accumulate more heavy metals and F, which raises significant food safety and health concerns. However, the mechanism behind this is not fully understood. Here, we report that tea plants responded to Al and F stresses by synthesizing and secreting OAs and altering profiles of amino acids, catechins, and caffeine in their roots. These organic compounds could form tea-plant mechanisms to tolerate lower pH and higher Al and F concentrations. Furthermore, high concentrations of Al and F stresses negatively affected the accumulation of tea secondary metabolites in young leaves, and thereby tea nutrient value. The young leaves of tea seedlings under Al and F stresses also tended to increase Al and F accumulation in young leaves but lower essential tea secondary metabolites, which challenged tea quality and safety. Comparisons of transcriptome data combined with metabolite profiling revealed that the corresponding metabolic gene expression supported and explained the metabolism changes in tea roots and young leaves via stresses from high concentrations of Al and F. The study provides new insight into Al- and F-stressed tea plants with regard to responsive metabolism changes and tolerance strategy establishment in tea plants and the impacts of Al/F stresses on metabolite compositions in young leaves used for making teas, which could influence tea nutritional value and food safety.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis.

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B. subtilis are still not fully explored. Here, a bottom-up approach is proposed for designing artificial trans-acting sRNAs. By engineering the intrinsic sRNA SR6, a minimized core scaffold structure consisting of an 8 bp stem, a 4 nt loop, and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity (83%). Moreover, we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli (above 80%). A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences, which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence. By applying these auto-designed sRNAs, we rapidly modified the cell morphology and biofilm formation, and regulated metabolic flux toward acetoin biosynthesis. This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B. subtilis and E. coli but also enhanced our understanding of trans-acting sRNAs.

Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves.

The tea plant is an economically important woody beverage crop. The unique taste of tea is evoked by certain metabolites, especially catechin esters, whereas their precise formation mechanism in different cell types remains unclear. Here, a fast protoplast isolation method was established and the transcriptional profiles of 16 977 single cells from 1st and 3rd leaves were investigated. We first identified 79 marker genes based on six isolated tissues and constructed a transcriptome atlas, mapped developmental trajectories and further delineated the distribution of different cell types during leaf differentiation and genes associated with cell fate transformation. Interestingly, eight differently expressed genes were found to co-exist at four branch points. Genes involved in the biosynthesis of certain metabolites showed cell- and development-specific characteristics. An unexpected catechin ester glycosyltransferase was characterized for the first time in plants by a gene co-expression network in mesophyll cells. Thus, the first single-cell transcriptional landscape in woody crop leave was reported and a novel metabolism pathway of catechin esters in plants was discovered.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters.

BACKGROUND: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. RESULTS: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. CONCLUSION: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Characterization of CsTSI in the Biosynthesis of Theanine in Tea Plants (Camellia sinensis).

Theanine is a unique major amino acid in tea plants responsible for umami taste and mental health benefits of tea. However, theanine biosynthesis and physiological role in tea plants are not fully understood. Here, we demonstrate that tea plant theanine synthetase is encoded by a glutamine synthetase gene CsTSI. The expression pattern of CsTSI is closely correlated with theanine and glutamine levels in various tissues. CsTSI transcripts were accumulated in root tip epidermal cells, pericycle and procambial cells, where CsTSI presents as a cytosolic protein. Ectopic expression of the gene in Arabidopsis led to greater glutamine and theanine production than controls when fed with ethylamine (EA). RNAi knockdown or overexpression of CsTSI in tea plant hairy roots reduced or enhanced theanine and glutamine contents, respectively, compared with controls. The CsTSI recombinant enzymes used glutamate as an acceptor and ammonium or EA as a donor to synthesize glutamine and theanine, respectively. CsTSI expression in tea roots responded to nitrogen supply and deprivation and was correlated with theanine contents. This study provides fresh insights into the molecular basis for the biosynthesis of theanine, which may facilitate the breeding of high-theanine tea plants for improving the nutritional benefit of tea.

Molecular Basis of the Distinct Metabolic Features in Shoot Tips and Roots of Tea Plants (Camellia sinensis): Characterization of MYB Regulator for Root Theanine Synthesis.

The physiological and metabolic differences between shoot tips and roots of tea plants are significant, and understanding them is required for improvement of tea quality and plant growth. A high-quality full-length transcriptome sequencing on tea plant roots and shoot tips by PacBio SMRT technology was done to gain a further understanding. Approximately 160699 and 166120 full-length transcripts were recovered in roots and shoots, respectively, including 31232 and 41068 novel isoforms and 16960 and 26029 alternative splicing (AS) isoforms. These supported 21699 full-length reads and 31232 and 41068 novel transcripts from root and shoot, respectively, including 1679 long noncoding RNAs (lncRNAs) and 2772 fusion transcripts, which significantly upgrade the Camellia sinensis genome annotation. The respective 6475 and 6981 transcripts in roots and shoots differ in 3'-untranslated regions. Meanwhile, extensive analyses of novel transcripts, ASs, and lncRNAs also revealed a large number of ASs and lincRNAs closely related to the regulation of characteristic secondary metabolites including catechins, theanine, and caffeine. Finally, a root-specific CsMYB6 was characterized to regulate theanine biosynthesis by genetic and molecular analyses. CsMYB6 directly bound to and activate the promoter of theanine synthetase gene (CsTSI). The study lays a foundation for the further investigation of metabolic genomics and regulation in tea plants.

Development of a Novel Biosensor-Driven Mutation and Selection System via in situ Growth of Corynebacterium crenatum for the Production of L-Arginine.

The high yield mutants require a high-throughput screening method to obtain them quickly. Here, we developed an L-arginine biosensor (ARG-Select) to obtain increased L-arginine producers among a large number of mutant strains. This biosensor was constructed by ArgR protein and argC promoter, and could provide the strain with the output of bacterial growth via the reporter gene sacB; strains with high L-arginine production could survive in 10% sucrose screening. To extend the screening limitation of 10% sucrose, the sensitivity of ArgR protein to L-arginine was decreased. Corynebacterium crenatum SYPA5-5 and its systems pathway engineered strain Cc6 were chosen as the original strains. This biosensor was employed, and L-arginine hyperproducing mutants were screened. Finally, the HArg1 and DArg36 mutants of C. crenatum SYPA5-5 and Cc6 could produce 56.7 and 95.5 g L-1 of L-arginine, respectively, which represent increases of 35.0 and 13.5%. These results demonstrate that the transcription factor-based biosensor could be applied in high yield strains selection as an effective high-throughput screening method.