Theanine metabolism and transport in tea plants (Camellia sinensis L.): advances and perspectives.

Theanine, a tea plant-specific non-proteinogenic amino acid, is the most abundant free amino acid in tea leaves. It is also one of the most important quality components of tea because it endows the "umami" taste, relaxation-promoting, and many other health benefits of tea infusion. Its content in tea leaves is directly correlated with the quality and price of green tea. Theanine biosynthesis primarily occurs in roots and is transported to new shoots in tea plants. Recently, great advances have been made in theanine metabolism and transport in tea plants. Along with the deciphering of the genomic sequences of tea plants, new genes in theanine metabolic pathway were discovered and functionally characterized. Theanine transporters were identified and were characterized on the affinity for: theanine, substrate specificity, spatiotemporal expression, and the role in theanine root-to-shoot transport. The mechanisms underlying the regulation of theanine accumulation by: cultivars, seasons, nutrients, and environmental factors are also being rapidly uncovered. Transcription factors were identified to be critical regulators of theanine biosynthesis. In this review, we summarize the progresses in theanine: biosynthesis, catabolism, and transport processes. We also discuss the future studies on theanine in tea plants, and application of the knowledge to crops to synthesize theanine to improve the health-promoting quality of non-tea crops.

Identification of key gene networks controlling polysaccharide accumulation in different tissues of Polygonatum cyrtonema Hua by integrating metabolic phenotypes and gene expression profiles.

Plant polysaccharides, a type of important bioactive compound, are involved in multiple plant defense mechanisms, and in particular polysaccharide-alleviated abiotic stress has been well studied. Polygonatum cyrtonema Hua (P. cyrtonema Hua) is a medicinal and edible perennial plant that is used in traditional Chinese medicine and is rich in polysaccharides. Previous studies suggested that sucrose might act as a precursor for polysaccharide biosynthesis. However, the role of sucrose metabolism and transport in mediating polysaccharide biosynthesis remains largely unknown in P. cyrtonema Hua. In this study, we investigated the contents of polysaccharides, sucrose, glucose, and fructose in the rhizome, stem, leaf, and flower tissues of P. cyrtonema Hua, and systemically identified the genes associated with the sucrose metabolism and transport and polysaccharide biosynthesis pathways. Our results showed that polysaccharides were mainly accumulated in rhizomes, leaves, and flowers. Besides, there was a positive correlation between sucrose and polysaccharide content, and a negative correlation between glucose and polysaccharide content in rhizome, stem, leaf, and flower tissues. Then, the transcriptomic analyses of different tissues were performed, and differentially expressed genes related to sucrose metabolism and transport, polysaccharide biosynthesis, and transcription factors were identified. The analyses of the gene expression patterns provided novel regulatory networks for the molecular basis of high accumulation of polysaccharides, especially in the rhizome tissue. Furthermore, our findings explored that polysaccharide accumulation was highly correlated with the expression levels of SUS, INV, SWEET, and PLST, which are mediated by bHLH, bZIP, ERF, ARF, C2H2, and other genes in different tissues of P. cyrtonema Hua. Herein, this study contributes to a comprehensive understanding of the transcriptional regulation of polysaccharide accumulation and provides information regarding valuable genes involved in the tolerance to abiotic stresses in P. cyrtonema Hua.

Transcriptome analysis provides insights into the molecular bases in response to different nitrogen forms-induced oxidative stress in tea plant roots (Camellia sinensis).

Previous studies have suggested that the maintenance of redox homeostasis is essential for plant growth. Here we investigated how redox homeostasis and signalling is modulated in response to different nitrogen (N) forms in tea plant roots. Our results showed that both N deficiency and nitrate (NO3-) can trigger the production of hydrogen peroxide and lipid peroxidation in roots. In contrast, these responses were not altered by NH4+. Further, N deficiency and NO3--triggered redox imbalance was re-established by increased of proanthocyanidins (PAs) and glutathione (GSH), as well as upregulation of representative antioxidant enzyme activities and genes. To further explore the molecular bases of these responses, comparative transcriptome analysis was performed, and redox homeostasis-associated differentially expressed genes (DEGs) were selected for bioinformatics analysis. Most of these genes were involved in the flavonoid biosynthesis, GSH metabolism and the antioxidant system, which was specifically altered by N deficiency or NO3-. Moreover, the interplay between H2O2 (generated by RBOH and Ndufab1) and hormones (including abscisic acid, auxin, cytokinin and ethylene) in response to different N forms was suggested. Collectively, the above findings contribute to an understanding of the underlying molecular mechanisms of redox homeostasis and signalling in alleviating oxidative stress in tea plant roots.

Transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root (Camellia sinensis L.).

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Overexpression of ethylene response factor ERF96 gene enhances selenium tolerance in Arabidopsis.

Ethylene response factors (ERFs) are involved in the regulation of plant responses to biotic and abiotic stresses. Here we provide evidence for a role of ERF96, a member of the ERF transcription factor group IX, in selenite tolerance in Arabidopsis. ERF96 gene was rapidly up-regulated in response to selenite stress. Overexpression of ERF96 enhanced Arabidopsis resistance to selenite stress, while ERF96-silenced plants demonstrated wild-type (WT) resistance to selenite. In addition, the overexpression plants had significantly lower selenium (Se) content in shoots when subjected to selenite stress. Further investigation indicated that overexpression of ERF96 reduced transcript levels of selenite/phosphate transporters PHT1;1 and PHT2;1, which influenced Arabidopsis Se uptake and allocation in the presence of selenite. Moreover, our experiments showed that overexpression of ERF96 enhanced Arabidopsis antioxidant activity. Under selenite stress, ERF96-overexpressing lines exhibited the significant increases in catalase (CAT) and glutathione peroxidase (GPX) activities as well as the glutathione (GSH) content, while had a decrease in reactive oxygen species (ROS) accumulation compared to WT. Taken together, our results demonstrate that ERF96 plays a positive role in the regulation of selenite tolerance in Arabidopsis.

The role of cytokinin in selenium stress response in Arabidopsis.

Cytokinins (CKs) regulate many developmental processes and environmental stress responses in plants. In this study, our data provide evidence that CK negatively regulates Arabidopsis selenium (Se) stress response. CK-deficient plant ipt1 3 5 7 exhibited enhanced Se tolerance which was abolished by exogenous benzylaminopurine (BA) application, while CK- receptor -deficient mutants ahk2 and ahk3 were sensitive to Se stress. Further investigation suggested that CK regulated Se tolerance of ipt1 3 5 7 through reduction of Se uptake and activation of metabolism detoxification, which had significantly lower transcriptions of high-affinity transporters PHT1;1, PHT1;8, PHT1;9 and the higher transcription of selenocysteine methyltransferase (SMT) respectively. Moreover, Se tolerance of ipt1 3 5 7 was associated with the enhanced antioxidant levels which had the higher catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities as well as the higher glutathione (GSH) content. On the other hand, loss-of-function mutations in single CK receptor genes could increase Se uptake and reactive oxygen species (ROS) accumulation, which caused Se sensitivity in ahk2 and ahk3 mutants. Taken together, these findings provide new insights to the role of CK in Se stress response in Arabidopsis.

Cytokinin is involved in TPS22-mediated selenium tolerance in Arabidopsis thaliana.

Background and Aims: Excess selenium (Se) is toxic to plants, but relatively little is known about the regulatory mechanism of plant Se tolerance. This study explored the role of the TPS22 gene in Se tolerance in Arabidopsis thaliana. Methods: Arabidopsis wild type and XVE mutant seeds were grown on half-strength MS media containing Na2SeO3 for screening of the Se-tolerant mutant tps22. The XVE T-DNA-tagged genomic sequence in tps22 was identified by TAIL-PCR. The TPS22 gene was transformed into the mutant tps22 and wild type plants using the flower infiltration method. Wild type, tps22 mutant and transgenic seedlings were cultivated on vertical plates for phenotype analysis, physiological index measurement and gene expression analysis. Key Results: We identified an Arabidopsis Se-tolerant mutant tps22 from the XVE pool lines, and cloned the gene which encodes the terpenoid synthase (TPS22). TPS22 was downregulated by Se stress, and loss-of-function of TPS22 resulted in decreased Se accumulation and enhanced Se tolerance; by contrast, overexpression of TPS22 showed similar traits to the wild type under Se stress. Further analysis revealed that TPS22 mediated Se tolerance through reduction of Se uptake and activation of metabolism detoxification, which decreased transcription of high-affinity transporters PHT1;1, PHT1;8 and PHT1;9 and significantly increased transcription of selenocysteine methyltransferase (SMT), respectively. Moreover, loss-of-function of TPS22 resulted in reduced cytokinin level and repression of cytokinin signalling components AHK3 and AHK4, and upregulation of ARR3, ARR15 and ARR16. Exogenous cytokinin increased transcription of PHT1;1, PHT2;1 and SMT and decreased Se tolerance of the tps22 mutant. In addition, enhanced Se resistance of the tps22 mutant was associated with glutathione (GSH). Conclusions: Se stress downregulated TPS22, which reduced endogenous cytokinin level, and then affected the key factors of Se uptake and metabolism detoxification. This cascade of events resulted in reduced Se accumulation and enhanced Se tolerance.

A role for APX1 gene in lead tolerance in Arabidopsis thaliana.

Lead (Pb) is a dangerous and widespread metal pollutant. Numerous studies have been made in understanding heavy metal detoxification and tolerance in plants, however, relatively few are known about the mechanisms involved in Pb stress response. In this study, we provide evidence for a novel role of APX1 gene in Pb tolerance in Arabidopsis. KO-APX1 mutants apx1-3 and apx1-4 showed more resistant than wild type, and the APX1-complementary COM1 restored the growth state of wild type in Pb stress. The two KO-APX1 mutants showed reduced Pb accumulation, which was accompanied by the activation of metal transporters PDR12 and ATM3 genes expression. In addition, glutathione (GSH), phytochelatin (PC) synthesis and related gene GSH1, GSH2, PCS1 and PCS2 expression were also increased in apx1-3 plants subjected to Pb stress. The more improvements in antioxidant enzymes glutathione peroxidase (GPX) and catalase (CAT) activities were found in the mutant apx1-3. Taken together, our results suggest that APX1 gene knockout results in enhanced Pb tolerance mainly through activating the expression of the ATP-bind cassette (ABC)-type transporters and at least partially through GSH -dependent PC synthesis pathway by coordinated control of gene expression.

Loss-of-function mutations in the APX1 gene result in enhanced selenium tolerance in Arabidopsis thaliana.

It is generally recognized that excess selenium (Se) has a negative effect on the growth and development of plants. Numerous studies have identified key genes involved in selenium tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. In this study, we isolated an Arabidopsis selenium-resistant mutant from the mutant XVE pool lines because of its increased root growth and fresh weight in Se stress, and cloned the gene, which encodes the cytosolic ascorbate peroxidase (APX1). Two other APX1 gene knockout allelic lines were also selenium resistant, and the APX1-complementary COM1 restored the growth state of wild type under Se stress. In addition, these APX1 allelic lines accumulated more Se than did wild-type plants when subjected to Se stress. Further analysis revealed that the APX1-mediated Se tolerance was associated, at least in part, with the enhanced activities of antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase. Moreover, enhanced Se resistance of the mutants was associated with glutathione (GSH), which had the higher expression level of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Our results provide genetic evidence indicating that loss-of-function of APX1 results in tolerance to Se stress.