Developmental and reproductive toxicity of a recombinant protein subunit COVID-19 vaccine (ZF2001) in rats.

ZF2001, a protein subunit vaccine against coronavirus disease 2019 (COVID-19), contains recombinant tandem repeat of dimeric receptor-binding domain (RBD) protein of the SARS-CoV-2 spike protein with an aluminium-based adjuvant. During the development of this vaccine, two nonclinical studies were conducted to evaluate female fertility, embryo-fetal development, and postnatal developmental toxicity in Sprague‒Dawley rats according to the ICH S5 (R3) guideline. In Study 1 (embryo-fetal developmental toxicity, EFD), 144 virgin female rats were randomly assigned into four groups and received three doses of vaccine (25 µg or 50 µg RBD protein/dose, containing the aluminium-based adjuvant), the aluminium-based adjuvant or a sodium chloride injection administered intramuscularly on days 21 and 7 prior to mating and on gestation day (GD) 6. In Study 2 (pre- and postnatal developmental toxicity, PPND), ZF2001 at a dose of 25 µg RBD protein/dose or sodium chloride injection was administered intramuscularly to female rats (n = 28 per group) 7 days prior to mating and on GD 6, GD 20 and postnatal day (PND) 10. There were no obvious adverse effects in dams, except for local injection site reactions related to the aluminium-based adjuvant (yellow nodular deposits in the interstitial muscle fibres). There were also no effects of ZF2001 on the mating performance, fertility or reproductive performance of parental females, embryo-fetal development, postnatal survival, growth, physical development, reflex ontogeny, behavioural and neurofunctional development, or reproductive performance of the offspring. The strong immune responses associated with binding and neutralising antibodies were both confirmed in dams and fetuses or offspring in these two studies. These results would support clinical trials or the use of ZF2001 in maternal immunisation campaigns, including those involving women with childbearing potential, regardless of pregnancy status.

Critical metabolic pathways and SAD/FADs, WRI1s, and DGATs cooperate for high-oleic acid oil production in developing oil tea (Camellia oleifera) seeds.

Oil tea trees produce high-quality edible oils with desirably high oleic acid (18:1) and low linoleic (18:2) and linolenic (18:3) fatty acid (FA) levels, but limited understanding of tea oil biosynthesis and regulation has become a significant obstacle for the breeding of high-yield and -quality oil tea varieties. By integrating metabolite and transcriptome analyses of developing oil tea seeds, we dissected the critical metabolic pathways, including glycolysis, fatty acid, and triacylglycerol (TAG) biosynthesis, as well as genes essential for tea seed oil production. Two plastidic stearoyl-acyl carrier protein desaturases (CoSAD1 and 2) and two endoplasmic reticulum-localized FA desaturases (CoFAD2 and 3) were functionally characterized as responsible for high 18:1 and low 18:2 and 18:3 proportions in tea oils. Two diacylglycerol O-acyltransferases (CoDGAT1 and 2) that may prefer to synthesize 18:1-TAG were functionally characterized and might be also important for high 18:1-TAG production. The highly expressed CoWRI1a and b were identified and characterized as activators of glycolysis and regulators of directing source carbon flux into FA biosynthesis in developing oil tea seeds. The upregulated CoSADs with downregulated CoFAD2 and CoFAD3 at the late seed developmental stages mainly accounted for high 18:1 levels. Two CoDGATs might be responsible for assembling TAGs with oleoyl acyl chains, whilst two CoWRI1s regulated carbons from parental sources, partitioning into oil production in oil tea embryo sinks. This study provides a deep understanding of the biosynthesis of tea seed oils and information on genes that may be used as molecular markers to breed oil tea varieties with higher oil yield and quality.

Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2.

Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

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.

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme.

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here, we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher order symmetry offers amenability to intricate regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced disorder-order transition paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for activity modulation, and sharpens our mechanistic understanding of the complex functional and regulatory properties of supramolecular enzymes.

Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients.

Antibody-dependent cellular cytotoxicity (ADCC) responses to viral infection are a form of antibody regulated immune responses mediated through the Fc fragment. Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered ADCC responses contributes to COVID-19 disease development is currently not well understood. To understand the potential correlation between ADCC responses and COVID-19 disease development, we analyzed the ADCC activity and neutralizing antibody response in 255 individuals ranging from asymptomatic to fatal infections over 1 year post disease. ADCC was elicited by 10 days post-infection, peaked by 11-20 days, and remained detectable until 400 days post-infection. In general, patients with severe disease had higher ADCC activities. Notably, patients who had severe disease and recovered had higher ADCC activities than patients who had severe disease and deceased. Importantly, ADCC activities were mediated by a diversity of epitopes in SARS-COV-2-infected mice and induced to comparable levels against SARS-CoV-2 variants of concern (VOCs) (B.1.1.7, B.1.351, and P.1) as that against the D614G mutant in human patients and vaccinated mice. Our study indicates anti-SARS-CoV-2 ADCC as a major trait of COVID-19 patients with various conditions, which can be applied to estimate the extra-neutralization level against COVID-19, especially lethal COVID-19.

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.

Genome-Wide Analysis of Serine Carboxypeptidase-Like Acyltransferase Gene Family for Evolution and Characterization of Enzymes Involved in the Biosynthesis of Galloylated Catechins in the Tea Plant (Camellia sinensis).

Tea (Camellia sinensis L.) leaves synthesize and concentrate a vast array of galloylated catechins (e.g., EGCG and ECG) and non-galloylated catechins (e.g., EGC, catechin, and epicatechin), together constituting 8%-24% of the dry leaf mass. Galloylated catechins account for a major portion of soluble catechins in tea leaves (up to 75%) and make a major contribution to the astringency and bitter taste of the green tea, and their pharmacological activity for human health. However, the catechin galloylation mechanism in tea plants is largely unknown at molecular levels. Previous studies indicated that glucosyltransferases and serine carboxypeptidase-like acyltransferases (SCPL) might be involved in the process. However, details about the roles of SCPLs in the biosynthesis of galloylated catechins remain to be elucidated. Here, we performed the genome-wide identification of SCPL genes in the tea plant genome. Several SCPLs were grouped into clade IA, which encompasses previously characterized SCPL-IA enzymes with an acylation function. Twenty-eight tea genes in this clade were differentially expressed in young leaves and vegetative buds. We characterized three SCPL-IA enzymes (CsSCPL11-IA, CsSCPL13-IA, CsSCPL14-IA) with galloylation activity toward epicatechins using recombinant enzymes. Not only the expression levels of these SCPLIA genes coincide with the accumulation of galloylated catechins in tea plants, but their recombinant enzymes also displayed ß-glucogallin:catechin galloyl acyltransferase activity. These findings provide the first insights into the identities of genes encoding glucogallin:catechin galloyl acyltransferases with an active role in the biosynthesis of galloylated catechins in tea plants.

Comparative Transcriptomic Analysis Reveals Regulatory Mechanisms of Theanine Synthesis in Tea (Camellia sinensis) and Oil Tea (Camellia oleifera) Plants.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (Camellia sinensis) and oil tea (Camellia oleifera) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.

The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future.

Metabolite profiling and transcriptomic analyses reveal an essential role of UVR8-mediated signal transduction pathway in regulating flavonoid biosynthesis in tea plants (Camellia sinensis) in response to shading.

BACKGROUND: Tea is the most popular nonalcoholic beverage worldwide for its pleasant characteristics and healthful properties. Catechins, theanine and caffeine are the major natural products in tea buds and leaves that determine tea qualities such as infusion colors, tastes and fragrances, as well as their health benefits. Shading is a traditional and effective practice to modify natural product accumulation and to enhance the tea quality in tea plantation. However, the mechanism underlying the shading effects is not fully understood. This study aims to explore the regulation of flavonoid biosynthesis in Camellia sinensis under shading by using both metabolomic and transcriptional analyses. RESULTS: While shading enhanced chlorophyll accumulation, major catechins, including C, EC, GC and EGC, decreased significantly in tea buds throughout the whole shading period. The reduction of catechins and flavonols were consistent with the simultaneous down-regulation of biosynthetic genes and TFs associated with flavonoid biosynthesis. Of 16 genes involved in the flavonoid biosynthetic pathway, F3'H and FLS significantly decreased throughout shading while the others (PAL, CHSs, DFR, ANS, ANR and LAR, etc.) temporally decreased in early or late shading stages. Gene co-expression cluster analysis suggested that a number of photoreceptors and potential genes involved in UV-B signal transductions (UVR8_L, HY5, COP1 and RUP1/2) showed decreasing expression patterns consistent with structural genes (F3'H, FLS, ANS, ANR, LAR, DFR and CHSs) and potential TFs (MYB4, MYB12, MYB14 and MYB111) involved in flavonoid biosynthesis, when compared with genes in the UV-A/blue and red/far-red light signal transductions. The KEGG enrichment and matrix correlation analyses also attributed the regulation of catechin biosynthesis to the UVR8-mediated signal transduction pathway. Further UV-B treatment in the controlled environment confirmed UV-B induction on flavonols and EGCG accumulation in tea leaves. CONCLUSIONS: We proposed that catechin biosynthesis in C. sinensis leaves is predominantly regulated by UV through the UVR8-mediated signal transduction pathway to MYB12/MYB4 downstream effectors, to modulate flavonoid accumulation. Our study provides new insights into our understanding of regulatory mechanisms for shading-enhanced tea quality.

De novo transcriptome sequencing and metabolite profiling analyses reveal the complex metabolic genes involved in the terpenoid biosynthesis in Blue Anise Sage (Salvia guaranitica L.).

Many terpenoid compounds have been extracted from different tissues of Salvia guaranitica. However, the molecular genetic basis of terpene biosynthesis pathways is virtually unknown. In this study, approximately 4 Gb of raw data were generated from the transcriptome of S. guaranitica leaves using Illumina HiSeq 2000 sequencing. After filtering and removing the adapter sequences from the raw data, the number of reads reached 32 million, comprising 186 million of high-quality nucleotide bases. A total of 61,400 unigenes were assembled de novo and annotated for establishing a valid database for studying terpenoid biosynthesis. We identified 267 unigenes that are putatively involved in terpenoid metabolism (including, 198 mevalonate and methyl-erythritol phosphate (MEP) pathways, terpenoid backbone biosynthesis genes and 69 terpene synthases genes). Moreover, three terpene synthase genes were studied for their functions in terpenoid biosynthesis by using transgenic Arabidopsis; most transgenic Arabidopsis plants expressing these terpene synthetic genes produced increased amounts of terpenoids compared with wild-type control. The combined data analyses from the transcriptome and metabolome provide new insights into our understanding of the complex metabolic genes in terpenoid-rich blue anise sage, and our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. guaranitica.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Transcriptome and metabolite analyses reveal the complex metabolic genes involved in volatile terpenoid biosynthesis in garden sage (Salvia officinalis).

A large number of terpenoid compounds have been extracted from different tissues of S. officinalis. However, the molecular genetic basis of terpene biosynthesis pathways is virtually unknown. In this study, approximately 6.6 Gb of raw data were generated from the transcriptome of S. officinalis leaves using Illumina HiSeq 2000 sequencing. After filtering and removing the adapter sequences from the raw data, the number of reads reached 21 million, comprising 98 million of high-quality nucleotide bases. 48,671 unigenes were assembled de novo and annotated for establishing a valid database for studying terpenoid biosynthesis. We identified 135 unigenes that are putatively involved in terpenoid metabolism, including 70 mevalonate and methyl-erythritol phosphate pathways, terpenoid backbone biosynthesis genes, and 65 terpene synthase genes. Moreover, five terpene synthase genes were studied for their functions in terpenoid biosynthesis by using transgenic tobacco; most transgenic tobacco plants expressing these terpene synthetic genes produced increased amounts of terpenoids compared with wild-type control. The combined data analyses from the transcriptome and metabolome provide new insights into our understanding of the complex metabolic genes in terpenoid-rich sage, and our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. officinalis.

Transport and transcriptional regulation of oil production in plants.

Triacylglycerol (TAG) serves as an energy reservoir and phospholipids as build blocks of biomembrane to support plant life. They also provide human with foods and nutrients. Multi-compartmentalized biosynthesis, trafficking or cross-membrane transport of lipid intermediates or precursors and their regulatory mechanisms are not fully understood. Recent progress has aided our understanding of how fatty acids (FAs) and phospholipids are transported between the chloroplast, the cytoplasm, and the endoplasmic reticulum (ER), and how the ins and outs of lipids take place in the peroxisome and other organelles for lipid metabolism and function. In addition, information regarding the transcriptional regulation network associated with FA and TAG biosynthesis has been further enriched. Recent breakthroughs made in lipid transport and transcriptional regulation has provided significant insights into our comprehensive understanding of plant lipid biology. This review attempts to highlight the recent progress made on lipid synthesis, transport, degradation, and their regulatory mechanisms. Metabolic engineering, based on these knowledge-powered technologies for production of edible oils or biofuels, is reviewed. The biotechnological application of metabolic enzymes, transcription factors and transporters, for oil production and composition improvement, are discussed in a broad context in order to provide a fresh scenario for researchers and to guide future research and applications.