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.

Theanine, a tea-plant-specific non-proteinogenic amino acid, is involved in the regulation of lateral root development in response to nitrogen status

Glutamine synthetase type I(GSI)-like proteins are proposed to mediate nitrogen signaling and developmental fate by synthesizing yet unidentified metabolites.Theanine,the most abundant non-proteinogenic amino acid in tea plants,is the first identified metabolite synthesized by a GSI-like protein(CsTSI)in a living system.However,the roles of theanine in nitrogen signaling and development are little understood.In this study we found that nitrogen deficiency significantly reduced theanine accumulation and increased lateral root development in tea plant seedlings.Exogenous theanine feeding significantly repressed lateral root development of seedlings of tea plants and the model plant Arabidopsis.The transcriptomic analysis revealed that the differentially expressed genes in the roots under theanine feeding were enriched in the apoplastic pathway and H2O_(2) metabolism.Consistently,theanine feeding reduced H2O_(2) levels in the roots.Importantly,when co-treated with H2O_(2),theanine abolished the promoting effect of H2O_(2) on lateral root development in both tea plant and Arabidopsis seedlings.The results of histochemical assays confirmed that theanine inhibited reactive oxygen species accumulation in the roots.Further transcriptomic analyses suggested the expression of genes encoding enzymes involved in H2O_(2) generation and scavenging was down-and upregulated by theanine,respectively.Moreover,the expression of genes involved in auxin metabolism and signaling,cell division,and cell expansion was also regulated by theanine.Collectively,these results suggested that CsTSI-synthesized theanine is likely involved in the regulation of lateral root development,via modulating H2O_(2) accumulation,in response to nitrogen levels in tea plants.This study also implied that the module consisting of GSI-like protein and theanine-like metabolite is probably conserved in regulating development in response to nitrogen status in plant species.

CsGDH2.1 negatively regulates theanine accumulation in late-spring tea plants (Camellia sinensis var. sinensis)

Theanine,a unique and the most abundant non-proteinogenic amino acid in tea plants,endows tea infusion with the umami taste and anti-stress effects.Its content in tea correlates highly with green tea quality.Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring.However,how the decrease is regulated is largely unknown.In a genetic screening,we observed that a yeast mutant,glutamate dehydrolase 2(gdh2),was hypersensitive to 40 mM theanine and accumulated more theanine.This result implied a role of CsGDH2s in theanine accumulation in tea plants.Therefore,we identified the two homologs of GDH2,CsGDH2.1 and CsGDH2.2,in tea plants.Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant.Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues.Importantly,CsGDH2.1 expression was low in early spring,and increased significantly in late spring,in the new shoots of tea plants.These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots.Moreover,the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity,and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants.These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring,and provides a functional gene for improving late-spring green tea quality.

CsWRKY13,a novel WRKY transcription factor of Camellia sinensis,involved in lignin biosynthesis and accumulation

Lignin is an aromatic polymer that provides the necessary mechanical strength for the transport of water and nutrients in higher plants.Lignin biosynthesis and accumulation affect growth and development of tea plants.The degree of lignification related to the tenderness of fresh tea leaves determines the quality of tea.WRKY transcription factors play central roles in plant development and physiological processes.However,the roles of WRKY transcription factors in lignin biosynthesis of tea plants remain unclear.In this study,a WRKY gene,CsWRKY13,was cloned from tea plant'Longjing 43'.The open reading frame(ORF)of CsWRKY13 gene was 708 bp,encoding 235 amino acids.Sequence analysis showed that CsWRKY13 contained a conserved WRKYGQK amino acid sequence and a zinc-finger-like motif CX4CX23HXH.Subcellular localization showed that CsWRKY13 was localized in the nucleus.The yeast trans-activation assay showed that CsWRKY13 had no transcriptional activity.Expression analysis showed that the CsWRKY13 gene was highly expressed in the stem.Overexpression of CsWRKY13 in Arabidopsis thaliana reduced lignin content and the expression levels of genes related to lignin biosynthesis in transgenic plants.Most flavonoids pathway related genes were significantly up-regulated.This study shows that CsWRKY13 might function as a negative regulator in regulation of lignin synthesis.