Real-time emulation of future global warming reveals realistic impacts on the phenological response and quality deterioration in rice.

Decreased production of crops due to climate change has been predicted scientifically. While climate-resilient crops are necessary to ensure food security and support sustainable agriculture, predicting crop growth under future global warming is challenging. Therefore, we aimed to assess the impact of realistic global warming conditions on rice cultivation. We developed a crop evaluation platform, the agro-environment (AE) emulator, which generates diverse environments by implementing the complexity of natural environmental fluctuations in customized, fully artificial lighting growth chambers. We confirmed that the environmental responsiveness of rice obtained in the fluctuation of artificial environments is similar to those exhibited in natural environments by validating our AE emulator using publicly available meteorological data from multiple years at the same location and multiple locations in the same year. Based on the representative concentration pathway, real-time emulation of severe global warming unveiled dramatic advances in the rice life cycle, accompanied by a 35% decrease in grain yield and an 85% increase in quality deterioration, which is higher than the recently reported projections. The transcriptome dynamism showed that increasing temperature and CO2 concentrations synergistically changed the expression of various genes and strengthened the induction of flowering, heat stress adaptation, and CO2 response genes. The predicted severe global warming greatly alters rice environmental adaptability and negatively impacts rice production. Our findings offer innovative applications of artificial environments and insights for enhancing varietal potential and cultivation methods in the future.

Deciphering transcriptomic signatures explaining the phenotypic plasticity of nonheading lettuce genotypes under artificial light conditions.

Elucidating the mechanisms and pathways involved in genotype-environment (G×E) interactions and phenotypic plasticity is critical for improving plant growth. Controlled environment agricultural systems allow growers to modulate the environment for particular genotypes. In this study, we evaluated the effects of interactions among 14 genotypes and four artificial light environments on leaf lettuce phenotypes and dissected the underlying molecular mechanism via transcriptome-based modeling. Variations in morphological traits and phytochemical concentrations in response to artificial light treatments revealed significant G×E interactions. The appropriate genotype and artificial light combinations for maximizing phenotypic expression were determined on the basis of a joint regression analysis and the additive main effect and multiplicative interaction model for these G×E interactions. Transcriptome-based regression modeling explained approximately 50%-90% of the G×E variations. Further analyzes indicated Red Lettuce Leaves 4 (RLL4) regulates UV-B and blue light signaling through the effects of the HY5-MBW pathway on flavonoid biosynthesis and contributes to natural variations in the light-responsive plasticity of lettuce traits. Our study represents an important step toward elucidating the phenotypic variations due to G×E interactions in nonheading lettuce under artificial light conditions.

Management of ureteral stenting for postrenal failure during pregnancy after ureteral reimplantation: a case report.

BACKGROUND: Vesicoureteral reflux is thought to predispose to urinary tract infection and renal scarring, and ureteral reimplantation in childhood remains the gold standard for its treatment. It has been reported that the risk of postrenal failure during pregnancy is increased among women with Politano-Leadbetter ureteral reimplantation. In previous case reports on patients with progressive hydronephrosis and renal failure during pregnancy after ureteral reimplantation, percutaneous nephrostomy was always required, so there has been no information about the clinical management of such patients by ureteral stenting. Here we report a patient with a history of bilateral ureteral reimplantation, in whom severe hydronephrosis during pregnancy was managed with ureteral stents. CASE PRESENTATION: A primigravida with severe hydronephrosis was referred to us at 29 weeks of gestation. Bilateral Politano-Leadbetter ureteral reimplantation had been performed at the age of 3 years. She was hospitalized immediately, and bilateral ureteral stents were successfully inserted. Post-obstructive diuresis occurred after the stents were placed. Urinary tract infection developed after removal of the urethral catheter 1 week later, but responded to antibiotic therapy and catheter replacement. Labor was induced at 39 weeks of gestation, with vaginal delivery of a healthy male infant. Both stents were found to have spontaneously migrated into the urethra after delivery. Repeat stenting under spinal anesthesia was required to improve postpartum symptoms of back pain and fever. Right distal ureteral obstruction persisted at 6 months after delivery and repeat ureteral reimplantation is planned. CONCLUSIONS: General obstetricians will not necessarily pay attention to a history of Politano-Leadbetter ureteral reimplantation, but these patients should undergo careful monitoring of renal function and urinary tract morphology during perinatal care. In the present case, ureteral stenting was effective for postrenal failure during pregnancy after ureteral reimplantation. If ureteral stenting is selected, attention should be paid to post-obstructive diuresis, infection, and stent migration.

[Modified Toyoda Method for Complete Duplex Ureters in Tubeless Cutaneous Ureterostomy : Report of a Case].

A 65-year-old man underwent radical cystectomy and cutaneous ureterostomy for bladder cancer. Because the patient had left complete duplex ureters,we modified the Toyoda method to construct the tubeless cutaneous ureterostomy with a unilateral stomal creation. The adhesive portion of both ureteral walls in duplex ureters was simultaneously cut approximately 5 mm from the distal end longitudinally,and the common sheath of the cut portion of the ureters was cut approximately 5 mm longitudinally in order to separate the two ureters. This procedure was repeated to make a"fish-mouth"aperture. Two opened ureteral walls were formed into the upper and lower plates of the Toyoda method stoma,respectively. To our knowledge,this is the first report of a surgical procedure of the tubeless cutaneous ureterostomy in complete duplex ureters.

[Clinical Study of Percutaneous Radiofrequency Ablation for Small Renal Tumor].

Percutaneous radiofrequency ablation (PRFA) has been applied as an option of minimally invasive treatment for small renal tumor. We retrospectively evaluated 5 patients with small renal tumor for whom PRFA was performed at our hospital. The average age was 69.6 years (range 45-86), average tumor diameter 20.0 mm (8-34), average preoperative glomerular filtration rate (eGFR) 66.8 ml/min/1.73m2 (42. 1-93.2), and follow-up period was 23.4 months (8-34). There were two minor complications (grade 1), including perirenal hematoma and pain at the probe insertion site in each patient. One patient had local recurrence 22 months after PRFA and the additional PRFA will be performed. PRFA could be a feasible, safe and effective therapy for small renal tumor.

[Questionnaire survey on medical care for male urethritis in community clinics in Shiga prefecture].

Six regional medical associations in Shiga prefecture agreed to cooperate in an investigation of medical care for male gonococcal and chlamydial urethritis. In June 2011, we sent a questionnaire to 372 medical offices in Shiga prefecture, and analyzed replies of respondents. Ten urologists and 175 non-urologists responded to the survey (response rate 49.7%). Among 185 physicians, 52 (10 urologists and 42 nonurologists) have treated male patients with gonococcal and chlamydial urethritis. More than 20% (42/175) of non-urological clinics are involved in the medical management. At initial diagnosis for sexually transmitted male urethritis, all urologists select the nucleic acid amplification method (100%), whereas many non-urologists do not (35%). For the treatment of chlamydial urethritis, non-urologists select levofloxacin (LVFX, 52.8%) rather than azithromycin (AZM, 22.0%), whereas urologists use AZM (78.0%) mostly but do not use LVFX (0%) (p = 0.023). For the treatment of gonococcal urethritis, non-urologists prefer oral new quinolones (53.1%) compared to urologists (25.0%) (p = 0. 74). For cure judgment of gonoccocal and chlamydial urethritis, many non-urologists rely on the improvement of subjective symptoms (50 and 47%), but urologists do not (10 and 0%) (p = 0.022 and 0.026, respectively). As for recognition of the clinical guideline for sexually transmitted disease, most urologists (90%) know it, but few non-urologists (13%) do (p < 0.001). We found that non-urological clinics make a great contribution to the medical treatment for male gonococcal and chlamydial urethritis in Shiga prefecture. It is important to standardize the medical care for sexually transmitted male urethritis by familiarizing non-urological practitioners with the clinical guideline.

Follicular thyroid cancer in children and adolescents: clinicopathologic features, long-term survival, and risk factors for recurrence.

Children and adolescents represent 1-1.5% of all patients with thyroid cancer (TC). The vast majority of TC in children and adolescents is papillary TC; follicular TC (FTC) is exceedingly rare. In this study, we evaluate the clinical and pathological features of FTC in children and adolescents. We also report the risk factors for post-operative tumor recurrence and the associated outcomes. Twenty children and adolescents (under 21 years old) with FTC have been treated and followed at Noguchi Thyroid Clinic and Hospital Foundation since 1946. All patients underwent surgery (lobectomy, 11; subtotal thyroidectomy, 8; and total thyroidectomy, 1), and 8 patients received postoperative external beam radiation therapy. The incidence of FTC in children and adolescents was 1.9% among all FTC patients treated in our hospital. Histopathology revealed vascular and capsular invasion in 9 and 20 patients, respectively. The tumor recurrence rate in FTC with vascular invasion is significantly higher than in those without it (p = 0.038). No other factors were significant. Patients with recurrences were treated with completion thyroidectomy and ¹³¹I radioactive iodine therapy. There were no significant differences in the rates of disease-free survival or cause-specific survival when pediatric/adolescent FTC patients were compared to adults with FTC. FTC is very rare among children and adolescents, but the outcomes are similar to those observed among adults. Vascular invasion is poor prognostic indicator in pediatric/adolescent FTC patients.

Genetic effects of Red Lettuce Leaf genes on red coloration in leaf lettuce under artificial lighting conditions.

Some cultivars of lettuce accumulate anthocyanins, which act as functional food ingredients. Leaf lettuce has been known to be erratic in exhibiting red color when grown under artificial light, and there is a need for cultivars that more stably exhibit red color in artificial light cultivation. In this study, we aimed to dissect the genetic architecture for red coloring in various leaf lettuce cultivars grown under artificial light. We investigated the genotype of Red Lettuce Leaf (RLL) genes in 133 leaf lettuce strains, some of which were obtained from publicly available resequencing data. By studying the allelic combination of RLL genes, we further analyzed the contribution of these genes to producing red coloring in leaf lettuce. From the quantification of phenolic compounds and corresponding transcriptome data, we revealed that gene expression level-dependent regulation of RLL1 (bHLH) and RLL2 (MYB) is the underlying mechanism conferring high anthocyanin accumulation in red leaf lettuce under artificial light cultivation. Our data suggest that different combinations of RLL genotypes cause quantitative differences in anthocyanin accumulation among cultivars, and some genotype combinations are more effective at producing red coloration even under artificial lighting.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

Potential of spectroscopic analyses for non-destructive estimation of tea quality-related metabolites in fresh new leaves.

Spectroscopic sensing provides physical and chemical information in a non-destructive and rapid manner. To develop non-destructive estimation methods of tea quality-related metabolites in fresh leaves, we estimated the contents of free amino acids, catechins, and caffeine in fresh tea leaves using visible to short-wave infrared hyperspectral reflectance data and machine learning algorithms. We acquired these data from approximately 200 new leaves with various status and then constructed the regression model in the combination of six spectral patterns with pre-processing and five algorithms. In most phenotypes, the combination of de-trending pre-processing and Cubist algorithms was robustly selected as the best combination in each round over 100 repetitions that were evaluated based on the ratio of performance to deviation (RPD) values. The mean RPD values were ranged from 1.1 to 2.7 and most of them were above the acceptable or accurate threshold (RPD = 1.4 or 2.0, respectively). Data-based sensitivity analysis identified the important hyperspectral regions around 1500 and 2000 nm. Present spectroscopic approaches indicate that most tea quality-related metabolites can be estimated non-destructively, and pre-processing techniques help to improve its accuracy.

Tissue-Dependent Variation Profiles of Tea Quality-Related Metabolites in New Shoots of Tea Accessions.

Several metabolites define tea quality in new tea shoots composed of leaf and stem. To improve tea quality for breeding, it is important to understand the tissue-dependent genetic mechanisms and metabolic network responsible for the profile of tea quality-related metabolites. We analyzed the volatiles and specialized metabolites as the tea quality-related metabolites in leaves and stems of new shoots in 30 tea accessions to understand the tissue variation and network between tea quality-related metabolites. Our results provided the tissue-dependent variation network in the tea quality-related metabolites, including volatiles in new leaves and stems in tea accessions. Each volatile content in tea accessions showed the coefficient of variation ranging from 58.7 to 221.9% and 54.2 to 318.3% in new leaves and new stems, respectively. The accumulation pattern of tea quality-related metabolites in new leaves and stems varied depending on the accession. When comparing tea genetic populations, the profile of tea quality-related metabolites of new leaves, but not new stems, was the key to distinguishing tea genetic populations by chemical indicators. We described the network between tea quality-related metabolites, especially the dense network in new leaves. These results also will provide the key information for metabolic engineering and the selection of breeding materials in tea plants based on the tea quality-related metabolites and aid in understanding their molecular mechanisms and network of metabolic variation.

Cesium uptake and translocation from tea cutting roots (Camellia sinensis L.).

To estimate the uptake of radiocesium (137Cs) by tea plant roots, 1-year-old rooted tea cuttings (Camellia sinensis L. cv. Yabukita) at the time of bud opening were cultivated hydroponically for 27 days in pots containing nutrient solutions with or without 137CsCl (600 Bq mL-1). Total 137Cs radioactivity of whole tea plants were 6.1 kBq g-1 dry weight. The plant/solution 137Cs transfer factors of different tissues were in the range of 2.6 (in mature leaves) to 28.2 mL g-1 dry weight (in roots), which were lower than those reported in wheat and spinach. In total, 69% of 137Cs remained in roots and 31% was transported from roots to shoots. The results indicated that 137Cs was preferentially translocated to new shoots, which are used for manufacturing tea, over mature leaves.

Dissection of hyperspectral reflectance to estimate nitrogen and chlorophyll contents in tea leaves based on machine learning algorithms.

Nondestructive techniques for estimating nitrogen (N) status are essential tools for optimizing N fertilization input and reducing the environmental impact of agricultural N management, especially in green tea cultivation, which is notably problematic. Previously, hyperspectral indices for chlorophyll (Chl) estimation, namely a green peak and red edge in the visible region, have been identified and used for N estimation because leaf N content closely related to Chl content in green leaves. Herein, datasets of N and Chl contents, and visible and near-infrared hyperspectral reflectance, derived from green leaves under various N nutrient conditions and albino yellow leaves were obtained. A regression model was then constructed using several machine learning algorithms and preprocessing techniques. Machine learning algorithms achieved high-performance models for N and Chl content, ensuring an accuracy threshold of 1.4 or 2.0 based on the ratio of performance to deviation values. Data-based sensitivity analysis through integration of the green and yellow leaves datasets identified clear differences in reflectance to estimate N and Chl contents, especially at 1325-1575 nm, suggesting an N content-specific region. These findings will enable the nondestructive estimation of leaf N content in tea plants and contribute advanced indices for nondestructive tracking of N status in crops.

Tissue ionome response to rhizosphere pH and aluminum in tea plants (Camellia sinensis L.), a species adapted to acidic soils.

The growth of tea plants (Camellia sinensis L.) is promoted by the presence of aluminum (Al), a beneficial element under acidic conditions, but the influence of rhizosphere pH on this interaction is not known. To understand the mechanisms underlying the adaptation to acidic rhizosphere conditions, we evaluated ionome profiles and the effect of pH on tea growth in hydroponic culture. The optimum pH for tea growth was around pH 4.2, and growth was inferior under a pH less than 3.8 or higher than 5.0. Under the optimum pH growth and Al accumulation were markedly stimulated by Al treatment. Al content and accumulation in new and mature leaves and new roots (the predominant tissues that accumulate minerals in tea plants) gradually declined with decrease in pH, especially in new roots. Ionome profiles drastically altered Al treatment, but changes were more pronounced in new roots than in new or mature leaves and did not depend on pH. Although the uptake of most cationic minerals in new roots was decreased by Al treatment, cationic mineral contents in new and mature leaves were not decreased by Al. In contrast to other plant species, the content and accumulation of manganese, despite it being a cationic nutrient, were significantly increased by Al treatment. These results indicated that one role of Al as a beneficial element was to maintain the shoot nutrient status by effectively utilizing Al-limited elements in the roots.

Genomic predictions and genome-wide association studies based on RAD-seq of quality-related metabolites for the genomics-assisted breeding of tea plants.

Effectively using genomic information greatly accelerates conventional breeding and applying it to long-lived crops promotes the conversion to genomic breeding. Because tea plants are bred using conventional methods, we evaluated the potential of genomic predictions (GPs) and genome-wide association studies (GWASs) for the genetic breeding of tea quality-related metabolites using genome-wide single nucleotide polymorphisms (SNPs) detected from restriction site-associated DNA sequencing of 150 tea accessions. The present GP, based on genome-wide SNPs, and six models produced moderate prediction accuracy values (r) for the levels of most catechins, represented by ( -)-epigallocatechin gallate (r = 0.32-0.41) and caffeine (r = 0.44-0.51), but low r values for free amino acids and chlorophylls. Integrated analysis of GWAS and GP detected potential candidate genes for each metabolite using 80-160 top-ranked SNPs that resulted in the maximum cumulative prediction value. Applying GPs and GWASs to tea accession traits will contribute to genomics-assisted tea breeding.

Integrated Metabolome and Transcriptome Analyses Reveal Etiolation-Induced Metabolic Changes Leading to High Amino Acid Contents in a Light-Sensitive Japanese Albino Tea Cultivar.

Plant albinism causes the etiolation of leaves because of factors such as deficiency of chloroplasts or chlorophylls. In general, albino tea leaves accumulate higher free amino acid (FAA) contents than do conventional green tea leaves. To explore the metabolic changes of etiolated leaves (EL) in the light-sensitive Japanese albino tea cultivar "Koganemidori," we performed integrated metabolome and transcriptome analyses by comparing EL with green leaves induced by bud-sport mutation (BM) or shading treatments (S-EL). Comparative omics analyses indicated that etiolation-induced molecular responses were independent of the light environment and were largely influenced by the etiolation itself. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and pathway analyses revealed the downregulation of genes involved in chloroplast development and chlorophyll biosynthesis and upregulation of protein degradation-related pathways, such as the ubiquitin-proteasome system and autophagy in EL. Metabolome analysis showed that most quantified FAAs in EL were highly accumulated compared with those in BM and S-EL. Genes involved in the tricarboxylic acid (TCA) cycle, nitrogen assimilation, and the urea cycle, including the drastically downregulated Arginase-1 homolog, which functions in nitrogen excretion for recycling, showed lower expression levels in EL. The high FAA contents in EL might result from the increased FAA pool and nitrogen source contributed by protein degradation, low N consumption, and stagnation of the urea cycle rather than through enhanced amino acid biosynthesis.

Phenotypic Markers Reflecting the Status of Overstressed Tea Plants Subjected to Repeated Shade Cultivation.

Shade cultivation is a traditional Japanese tea cultivation method in which the shoot buds are shaded for several weeks. This technique is increasingly used for green tea production because it produces tea of high quality (as indicated by umami and nutritional content) and commands high prices. However, given that shaded tea plants are grown under low-light stress, concerns exist regarding damage to tea plants caused by repeated shade cultivation. To understand basic physiological responses and accumulative changes in photosynthetic ability and metabolites of tea plants subjected to repeated shading, we performed a pot experiment on immature tea plants grown in a growth chamber subjected to repeated shading treatments. The results demonstrated that shade cultivation caused a decrease in non-structural carbohydrate content and an increase of several degrees in leaf surface temperature, reflecting transpiration through the leaf stomata, as a result of a reduction in photosynthetic ability. An increase of several degrees in canopy temperature and a reduction in photosynthetic ability in the field in the mid-summer season was also observed in overstressed tea plants subjected to repeated shading. Metabolomic analysis identified several candidate biomarkers, such as citrulline and glycine betaine, that were significantly changed in individuals affected by shade cultivation. These physiological changes may be an indicator of the stress status of tea plants grown under repeated shade cultivation.

Successful treatment of anaplastic thyroid carcinoma with a combination of oral valproic acid, chemotherapy, radiation and surgery.

Anaplastic thyroid carcinoma (ATC) is the most aggressive of thyroid cancers whose treatment is not yet established and mortality is extremely high. Recent in vitro studies have shown that valproic acid (VA), a newly identified histone deacetilase (HDAC) inhibitor, induces apoptosis, modulates differentiation gene expression of thyroid tumors and enhances the sensitivity of anaplastic cancer cell lines to doxorubicin. We report a case of successful treatment of anaplastic thyroid carcinoma with a combination of oral valproic acid, chemotherapy consisting of cisplatin and doxorubicin, external and intra-operative radiation and surgery. Tumor volume decreased by 50.7% under CT measurement and 44.6% under sonogram measurement over the course of the treatment. No significant rebound of tumor size was observed between each cycle of chemotherapy. Serial cytology performed via fine needle aspiration (FNA) presented a rapidly changing profile of cell types, starting with anaplastic and proceeding through increasingly well differentiated presentations. Only microscopic remnants of ATC cells were found in the histological examination of the resected thyroid. Ga scintigraphy and whole body PET scan six months after surgery revealed no evidence of recurrence or metastasis. As of Nov. 22, 2008, the patient is alive and disease free two years after diagnosis.

Analyses of single nucleotide polymorphisms identified by ddRAD-seq reveal genetic structure of tea germplasm and Japanese landraces for tea breeding.

To obtain genetic information about the germplasm of tea (Camellia sinensis L.) in Japan, 167 accessions including 138 var. sinensis (96 Japanese var. sinensis and 42 exotic var. sinensis) and 29 Assam hybrids were analyzed using single nucleotide polymorphisms (SNPs) markers identified by double-digest restriction-site-associated DNA sequencing (ddRAD-seq) analysis. Approximately 10,000 SNPs were identified by ddRAD-seq and were mapped across the whole genome. The 167 tea accessions were classified into three genetic subgroups: (1) Japanese var. sinensis; (2) Japanese and exotic var. sinensis; (3) Assam hybrids and exotic var. sinensis. Leaf morphology varied widely within each genetic subgroups. The 96 Japanese var. sinensis were classified into four genetic subgroups as follows; two subgroups of Shizuoka (the largest tea production region) landraces, Uji (most ancient tea production region) landraces, and the pedigree of 'Yabukita', the leading green tea cultivar in Japan. These results indicated that the SNP markers obtained from ddRAD-seq are a useful tool to investigate the geographical background and breeding history of Japanese tea. This genetic information revealed the ancestral admixture situation of the 'Yabukita' pedigree, and showed that the genome structure of 'Yabukita' is clearly different from those of other Japanese accessions.

Quantitative validation of nicotine production in tea (Camellia sinensis L.).

Endogenous nicotine was confirmed to be present in tea plants (Camellia sinensis L.) by liquid chromatography-tandem mass spectrometry of tea samples from tea-producing regions in six Asian countries. All samples contained nicotine (0.011-0.694 µg g-1 dry weight). Nicotine contents remained constant during manufacturing of green, oolong and black teas, implying that nicotine is stable against heating, drying, enzymatic oxidation and mechanical damage during processing. Flower buds and seeds of cultivar Yabukita also contained nicotine (0.030-0.041 µg g-1 dry weight). A comparison of two cultivars revealed that higher nicotine contents were found in the black tea cultivar Benifuki. All plant parts of hydroponic Yabukita contained nicotine (0.003-0.013 µg g-1 dry weight). Tea cells cultured in B5 medium as well as roots and stems of tea seedlings contained nicotine levels similar to those of new leaves from field-grown plants. Although the levels of endogenous nicotine in tea plants are extremely low and sample contamination cannot be discounted, these levels exceed the maximum acceptable limit in Japan (0.01 µg g-1 dry weight).