PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Haem Oxygenase 1 is a potential target for creating etiolated/albino tea plants (Camellia sinensis) with high theanine accumulation.

Theanine content is highly correlated with sensory quality and health benefits of tea infusion. The tender shoots of etiolated and albino tea plants contain higher theanine than the normal green tea plants and are valuable materials for high quality green tea processing. However, why these etiolated or albino tea plants can highly accumulate theanine is largely unknown. In this study, we observed an Arabidopsis etiolated mutant hy1-100 (mutation in Haem Oxygenase 1, HO1) that accumulated higher levels of glutamine (an analog of theanine). We therefore identified CsHO1 in tea plants and found CsHO1 is conserved in amino acid sequences and subcellular localization with its homologs in other plants. Importantly, CsHO1 expression in the new shoots was much lower in an etiolated tea plants 'Huangkui' and an albino tea plant 'Huangshan Baicha' than that in normal green tea plants. The expression levels of CsHO1 were negatively correlated with theanine contents in these green, etiolated and albino shoots. Moreover, CsHO1 expression levels in various organs and different time points were also negatively correlated with theanine accumulation. The hy1-100 was hypersensitive to high levels of theanine and accumulated more theanine under theanine feeding, and these phenotypes were rescued by the expression of CsHO1 in this mutant. Transient knockdown CsHO1 expression in the new shoots of tea plant using antisense oligonucleotides (asODN) increased theanine accumulation. Collectively, these results demonstrated CsHO1 negatively regulates theanine accumulation in tea plants, and that low expression CsHO1 likely contributes to the theanine accumulation in etiolated/albino tea plants.

Long-read sequencing: An effective method for genetic analysis of CYP21A2 variation in congenital adrenal hyperplasia.

BACKGROUND: The sequence similarity between CYP21A2 gene and its inactive pseudogene CYP21A1P, and copy number variation (CNV) caused by unequal crossover, make it challenging to characterize the CYP21A2 gene through traditional methods. This study aimed to evaluate the clinical utility of the long-read sequencing (LRS) method in carrier screening and genetic diagnosis of congenital adrenal hyperplasia (CAH) by comparing the efficiency of the LRS method with the conventional multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing approaches in CYP21A2 analysis. METHODS: In a retrospective study, full sequence analysis of the CYP21A2 and CYP21A1P was performed for three pedigrees through long-range locus-specific PCR followed by LRS based on the Pacific Biosciences (PacBio, California, USA) single-molecule real-time (SMRT) platform, and the results were compared with those obtained from next-generation sequencing (NGS)-based whole exome sequencing (WES) and the traditional methods of MLPA plus Sanger sequencing. RESULTS: The LRS method successfully identified seven CYP21A2 variants, including three single nucleotide variants (NM_000500.9:c.1451G > C p.(Arg484Pro), c.293-13A/C > G (IVS2-13A/C > G), c.518 T > A p.(Ile173Asn)), one 111-bp polynucleotide insertion, one set of 3'URT variants (NM_000500.9:c.*368 T > C, c.*390A > G, c.*440C > T, c.*443 T > C) and two types of chimeric genes and straightforwardly depicted the inheritance patterns of these variants within families. Moreover, the LRS method enabled us to determine the cis-trans configuration of multiple variants in one assay, without the need to analyze additional family samples. Compared with traditional methods, this LRS method can achieve a precise, comprehensive and intuitive result in the genetic diagnosis of 21-hydroxylase deficiency (21-OHD). CONCLUSION: The LRS method is comprehensive in CYP21A2 analysis and intuitive in result presentation, which holds substantial promise in clinical application as a crucial tool for carrier screening and genetic diagnosis of CAH.

Prmt5 promotes ciliated cell specification of airway epithelial progenitors via transcriptional inhibition of Tp63.

The epithelium of the pulmonary airway is composed of several distinct cell types that differentiate from common progenitor cells to provide defense against environmental insults. Epigenetic mechanisms regulating lineage differentiation of airway epithelial progenitors remain poorly understood. Protein arginine methyltransferase 5 (Prmt5) is a predominant type II arginine methyltransferase that methylates >85% of symmetric arginine residues. Here, we provide evidence for the function of Prmt5 in promoting ciliated cell fate specification of airway epithelial progenitors. We show that lung epithelial-specific deletion of Prmt5 resulted in a complete loss of ciliated cells, an increased number of basal cells, and ecotopic-expressed Tp63-Krt5+ putative cells in the proximal airway. We further identified that transcription factor Tp63 is a direct target of Prmt5, and Prmt5 inhibited Tp63 transcription expression through H4R3 symmetric dimethylation (H4R3sme2). Moreover, inhibition of Tp63 expression in Prmt5-deficient tracheal progenitors could partially restore the ciliated cell deficient phenotype. Together, our data support a model where Prmt5-mediated H4R3sme2 represses Tp63 expression to promote ciliated cell fate specification of airway progenitors.

PRMT5 determines the pattern of polyploidization and prevents liver from cirrhosis and carcinogenesis.

Human hepatocellular carcinoma (HCC) occurs almost exclusively in cirrhotic livers. Here, we report that hepatic loss of protein arginine methyltransferase 5 (PRMT5) in mice is sufficient to cause cirrhosis and HCC in a clinically relevant way. Furthermore, pathological polyploidization induced by hepatic loss of PRMT5 promotes liver cirrhosis and hepatic tumorigenesis in aged liver. The loss of PRMT5 leads to hyper-accumulation of P21 and endoreplication-dependent formation of pathological mono-nuclear polyploid hepatocytes. PRMT5 and symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) directly associate with chromatin of P21 to suppress its transcription. More importantly, loss of P21 rescues the pathological mono-nuclear polyploidy and prevents PRMT5-deficiency-induced liver cirrhosis and HCC. Thus, our results indicate that PRMT5-mediated symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) is crucial for preventing pathological polyploidization, liver cirrhosis and tumorigenesis in mouse liver.

Generation of GM130 Conditional Knockout Mouse.

The Golgi apparatus is a common and highly dynamic organelle in eukaryotic cells. It plays an important role in secretory trafficking and cargo modifications. Increasing evidence suggests that structural changes and functional disorders of the Golgi apparatus are involved in many human diseases, but whether Golgi dysfunction is a causal factor in regard to the progression of these diseases remains unknown. GM130 has been postulated to play roles in Golgi stack formation and vesicular transport based on studies on cultured cells and in vitro reconstitutions. To define the role of GM130 in animal, a GM130 knockout mouse has recently been created. Based on the principle of homologous recombination, the GM130 conditional knockout mouse model was established through gene targeting, stem cell screening, and blastocyst injection. Such model has been successfully applied for studies of physiological functions of GM130 and Golgi apparatus at the cellular and animal levels.

Single-cell chromatin accessibility identifies enhancer networks driving gene expression during spinal cord development in mouse.

Spinal cord development is precisely orchestrated by spatiotemporal gene regulatory programs. However, the underlying epigenetic mechanisms remain largely elusive. Here, we profiled single-cell chromatin accessibility landscapes in mouse neural tubes spanning embryonic days 9.5-13.5. We identified neuronal-cell-cluster-specific cis-regulatory elements in neural progenitors and neurons. Furthermore, we applied a novel computational method, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression data and identify the hub enhancers within enhancer networks. It was experimentally validated in vivo for Atoh1 that knockout of the hub enhancers, but not the non-hub enhancers, markedly decreased Atoh1 expression and reduced dp1/dI1 cells. Together, our work provides insights into the epigenetic regulation of spinal cord development and a proof-of-concept demonstration of enhancer networks as a general mechanism in transcriptional regulation.

Loss of PRMT7 reprograms glycine metabolism to selectively eradicate leukemia stem cells in CML.

Our group has reported previously on the role of various members of the protein arginine methyltransferase (PRMT) family, which are involved in epigenetic regulation, in the progression of leukemia. Here, we explored the role of PRMT7, given its unique function within the PRMT family, in the maintenance of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Genetic loss of Prmt7, and the development and testing of a small-molecule specific inhibitor of PRMT7, showed that targeting PRMT7 delayed leukemia development and impaired self-renewal of LSCs in a CML mouse model and in primary CML CD34+ cells from humans without affecting normal hematopoiesis. Mechanistically, loss of PRMT7 resulted in reduced expressions of glycine decarboxylase, leading to the reprograming of glycine metabolism to generate methylglyoxal, which is detrimental to LSCs. These findings link histone arginine methylation with glycine metabolism, while suggesting PRMT7 as a potential therapeutic target for the eradication of LSCs in CML.

GM130 regulates pulmonary surfactant protein secretion in alveolar type II cells.

Pulmonary surfactant is a lipid-protein complex secreted by alveolar type II epithelial cells and is essential for the maintenance of the delicate structure of mammalian alveoli to promote efficient gas exchange across the air-liquid barrier. The Golgi apparatus plays an important role in pulmonary surfactant modification and secretory trafficking. However, the physiological function of the Golgi apparatus in the transport of pulmonary surfactants is unclear. In the present study, deletion of GM130, which encodes for a matrix protein of the cis-Golgi cisternae, was shown to induce the disruption of the Golgi structure leading to impaired secretion of lung surfactant proteins and lipids. Specifically, the results of in vitro and in vivo analysis indicated that the loss of GM130 resulted in trapping of Sftpa in the endoplasmic reticulum, Sftpb and Sftpc accumulation in the Golgi apparatus, and an increase in the compensatory secretion of Sftpd. Moreover, global and epithelial-specific GM130 knockout in mice resulted in an enlargement of alveolar airspace and an increase in alveolar epithelial autophagy; however, surfactant repletion partially rescued the enlarged airspace defects in GM130-deficient mice. Therefore, our results demonstrate that GM130 and the mammalian Golgi apparatus play a critical role in the control of surfactant protein secretion in pulmonary epithelial cells.

PRMT7 targets of Foxm1 controls alveolar myofibroblast proliferation and differentiation during alveologenesis.

Although aberrant alveolar myofibroblasts (AMYFs) proliferation and differentiation are often associated with abnormal lung development and diseases, such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), epigenetic mechanisms regulating proliferation and differentiation of AMYFs remain poorly understood. Protein arginine methyltransferase 7 (PRMT7) is the only reported type III enzyme responsible for monomethylation of arginine residue on both histone and nonhistone substrates. Here we provide evidence for PRMT7's function in regulating AMYFs proliferation and differentiation during lung alveologenesis. In PRMT7-deficient mice, we found reduced AMYFs proliferation and differentiation, abnormal elastin deposition, and failure of alveolar septum formation. We further shown that oncogene forkhead box M1 (Foxm1) is a direct target of PRMT7 and that PRMT7-catalyzed monomethylation at histone H4 arginine 3 (H4R3me1) directly associate with chromatin of Foxm1 to activate its transcription, and thereby regulate of cell cycle-related genes to inhibit AMYFs proliferation and differentiation. Overexpression of Foxm1 in isolated myofibroblasts (MYFs) significantly rescued PRMT7-deficiency-induced cell proliferation and differentiation defects. Thus, our results reveal a novel epigenetic mechanism through which PRMT7-mediated histone arginine monomethylation activates Foxm1 transcriptional expression to regulate AMYFs proliferation and differentiation during lung alveologenesis and may represent a potential target for intervention in pulmonary diseases.

The tea plant CsLHT1 and CsLHT6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations.

Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids inorganic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When 15N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1, CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22∆10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H+-dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15N-glutamate and 15N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown inorganic tea plantations.

PRMT5 regulates ovarian follicle development by facilitating Wt1 translation.

Protein arginine methyltransferase 5 (Prmt5) is the major type II enzyme responsible for symmetric dimethylation of arginine. Here, we found that PRMT5 was expressed at high level in ovarian granulosa cells of growing follicles. Inactivation of Prmt5 in granulosa cells resulted in aberrant follicle development and female infertility. In Prmt5-knockout mice, follicle development was arrested with disorganized granulosa cells in which WT1 expression was dramatically reduced and the expression of steroidogenesis-related genes was significantly increased. The premature differentiated granulosa cells were detached from oocytes and follicle structure was disrupted. Mechanism studies revealed that Wt1 expression was regulated by PRMT5 at the protein level. PRMT5 facilitated IRES-dependent translation of Wt1 mRNA by methylating HnRNPA1. Moreover, the upregulation of steroidogenic genes in Prmt5-deficient granulosa cells was repressed by Wt1 overexpression. These results demonstrate that PRMT5 participates in granulosa cell lineage maintenance by inducing Wt1 expression. Our study uncovers a new role of post-translational arginine methylation in granulosa cell differentiation and follicle development.

Infertility in women can be caused by many factors, such as defects in the ovaries. An important part of the ovaries for fertility are internal structures called follicles, which house early forms of egg cells. A follicle grows and develops until the egg is finally released from the ovary into the fallopian tube, where the egg can then be fertilised. In the follicle, an egg is surrounded by other types of cells, such as granulosa cells. The egg and neighbouring cells must maintain healthy contacts with each other, otherwise the follicle can stop growing and developing, potentially causing infertility. The development of a follicle depends on an array of proteins. For example, the transcription factor WT1 controls protein levels by activating other genes and their proteins and is produced in high numbers by granulosa cells at the beginning of follicle development. Although WT1 levels dip towards the later stages of follicle development, insufficient levels can lead to defects. So far, it has been unclear how levels of WT1in granulose cells are regulated. Chen, Dong et al. studied mouse follicles to reveal more about the role of WT1 in follicle development. The researchers measured protein levels in mouse granulosa cells as the follicles developed, and discovered elevated levels of PRMT5, a protein needed for egg cells to form and survive in the follicles. Blocking granulosa cells from producing PRMT5 led to abnormal follicles and infertility in mice. Moreover, mice that had been engineered to lack PRMT5 developed abnormal follicles, where the egg and surrounding granulosa cells were not attached to each other, and the granulosa cells had low levels of WT1. Further experiments revealed that PRMT5 controlled WT1 levels by adding small molecules called methyl groups to another regulatory protein called HnRNPA1. The addition of methyl groups to genes or their proteins is an important modification that takes place in many processes within a cell. Chen, Dong et al. reveal that this activity also plays a key role in maintaining healthy follicle development in mice, and that PRMT5 is necessary for controlling WT1. Identifying all of the intricate mechanism involved in regulating follicle development is important for finding ways to combat infertility.

A multi-omics investigation of the composition and function of extracellular vesicles along the temporal trajectory of COVID-19.

Exosomes represent a subtype of extracellular vesicle that is released through retrograde transport and fusion of multivesicular bodies with the plasma membrane1. Although no perfect methodologies currently exist for the high-throughput, unbiased isolation of pure plasma exosomes2,3, investigation of exosome-enriched plasma fractions of extracellular vesicles can confer a glimpse into the endocytic pathway on a systems level. Here we conduct high-coverage lipidomics with an emphasis on sterols and oxysterols, and proteomic analyses of exosome-enriched extracellular vesicles (EVs hereafter) from patients at different temporal stages of COVID-19, including the presymptomatic, hyperinflammatory, resolution and convalescent phases. Our study highlights dysregulated raft lipid metabolism that underlies changes in EV lipid membrane anisotropy that alter the exosomal localization of presenilin-1 (PS-1) in the hyperinflammatory phase. We also show in vitro that EVs from different temporal phases trigger distinct metabolic and transcriptional responses in recipient cells, including in alveolar epithelial cells, which denote the primary site of infection, and liver hepatocytes, which represent a distal secondary site. In comparison to the hyperinflammatory phase, EVs from the resolution phase induce opposing effects on eukaryotic translation and Notch signalling. Our results provide insights into cellular lipid metabolism and inter-tissue crosstalk at different stages of COVID-19 and are a resource to increase our understanding of metabolic dysregulation in COVID-19.

Shading Promoted Theanine Biosynthesis in the Roots and Allocation in the Shoots of the Tea Plant (Camellia sinensis L.) Cultivar Shuchazao.

Shading was thought as an effective approach to increase theanine in harvested tea shoots. Previous studies offered conflicting findings, perhaps since the integration of theanine metabolism and transport in different tissues was not considered. Theanine is synthesized primarily in the roots and is then transported, via the vascular system, to new vegetative tissues. Here, we found that theanine increased in the stem, was reduced in the leaf, and remained stable in the roots, under shading conditions. Notably, in tea roots, shading significantly increased ethylamine and activated the theanine biosynthesis pathway and theanine transporter genes. Furthermore, shading significantly increased the expression of theanine transporter genes, CsAAP2/4/5/8, in the stem, while decreasing the expression of CsAAP1/2/4/5/6 in the leaf, in accordance with shading effects on theanine levels in these tissues. These findings reveal that shading of tea plants promotes theanine biosynthesis and allocation in different tissues, processes which appear to involve the theanine biosynthesis pathway enzymes and AAP family of theanine transporters.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage.

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7-/- mice at E11.5. We also find that the size of testes is reduced in Prmt7-/- mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-ß singling pathway is significantly changed in germ cells of Prmt7-/- mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1.

Many animal viral proteins, e.g., Vpr of HIV-1, disrupt host mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1. However, it is unknown whether plant viruses may use this mechanism in their pathogenesis. Here, we report that the 17K protein, encoded by barley yellow dwarf viruses and related poleroviruses, delays G2/M transition and disrupts mitosis in both host (barley) and nonhost (fission yeast, Arabidopsis thaliana, and tobacco) cells through interrupting the function of Wee1-Cdc25-CDKA/Cdc2 via direct protein-protein interactions and alteration of CDKA/Cdc2 phosphorylation. When ectopically expressed, 17K disrupts the mitosis of cultured human cells, and HIV-1 Vpr inhibits plant cell growth. Furthermore, 17K and Vpr share similar secondary structural feature and common amino acid residues required for interacting with plant CDKA. Thus, our work reveals a distinct class of mitosis regulators that are conserved between plant and animal viruses and play active roles in viral pathogenesis.

Theanine transporters are involved in nitrogen deficiency response in tea plant (Camellia sinensis L.).

Nitrogen in soil directly influences the production and quality of tea. However, high nitrogen application in tea plantation leads to soil acidification and environmental pollution. Studies in model plants showed that plasma membrane localized amino acid transporter can regulate the distribution of amino acids to enhance nitrogen use efficiency. Our recent study identified six CsAAPs as transporters for theanine, a unique and most abundant non-proteinaceous amino acid in tea plant. In this work, we found these theanine transporters can also transport Glutamine, Glutamate, aspartate, alanine and γ-aminobutyric acid. Tissue-specific expression analyses showed that CsAAP1, CsAAP5 and CsAAP6 mainly expressed in leaves, CsAAP8 in root, CsAAP4 and CsAAP2 in stem. Furthermore, the expression of these CsAAPs was induced by nitrogen deficiency in a tissue-specific manner. Subcellular localization analyses showed that CsAAP1, CsAAP2 and CsAAP6 location were in the plasma membrane and endoplasmic reticulum. Taken together, these results suggested theanine transporters are involved in nitrogen deficiency response probably by mediating amino acid transport from roots to new shoots and from source to sink tissues in tea plants.

Theanine transporters identified in tea plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.