Cytosolic Nudix Hydrolase 1 Is Involved in Geranyl ß-Primeveroside Production in Tea.

Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl ß-primeveroside were dramatically higher than those of geranyl ß-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl ß-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl ß-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl ß-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl ß-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar "Shucha Zao," indicating that CsNUDX1-cyto is important for geranyl ß-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl ß-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl ß-primeveroside in plants was also discussed.

UV-B Induces Distinct Transcriptional Re-programing in UVR8-Signal Transduction, Flavonoid, and Terpenoids Pathways in Camellia sinensis.

Plants are known to respond to Ultraviolet-B radiation (UV-B: 280-320 nm) by generating phenolic metabolites which absorbs UV-B light. Phenolics are extraordinarily abundant in Camellia sinensis leaves and are considered, together with pleasant volatile terpenoids, as primary flavor determinants in tea beverages. In this study, we focused on the effects of UV-B exposure (at 35 µW cm-2 for 0, 0.5, 2, and 8 h) on tea transcriptional and metabolic alterations, specifically related to tea flavor metabolite production. Out of 34,737 unigenes, a total of 18,081 differentially expressed genes (DEGs) due to UV-B treatments were identified. Additionally, the phenylpropanoid pathway was found as one of the most significantly UV-B affected top 20 KEGG pathways while flavonoid and monoterpenoid pathway-related genes were enhanced at 0.5 h. In the UVR8-signal transduction pathway, UVR8 was suppressed at both short and long exposure of UV-B with genes downstream differentially expressed. Divergent expression of MYB4 at different treatments could have differentially altered structural and regulatory genes upstream of flavonoid biosynthesis pathways. Suppression of MYB4-1&3 at 0.5 h could have led to the up-regulation of structural CCOAOMT-1&2, HST-1&2, DFR-4, ANR-2, and LAR-1&3 genes resulting in accumulation of specialized metabolites at a shorter duration of UV-B exposure. Specialized metabolite profiling revealed the correlated alterations in the abundances of catechins and some volatile terpenoids in all the treatments with significant accumulation of specialized metabolites at 0.5 h treatment. A significant increase in specialized metabolites at 0.5 h treatment and no significant alteration observed at longer UVB treatment suggested that shorter exposure to UV-B led to different display in gene expression and accumulation of specialized metabolites in tea shoots in response to UV-B stress. Taken together, our results indicated that the UV-B treatment applied in this study differentially altered the UVR8-signal transduction, flavonoid and terpenoid pathways at transcriptional and metabolic levels in tea plants. Our results show strong potential for UV-B application in flavor improvement in tea at the industrial level.

Analysis of Terpene Synthase Family Genes in Camellia sinensis with an Emphasis on Abiotic Stress Conditions.

For a better understanding terpenoid volatile production in Camellia sinensis, global terpenoid synthase gene (TPS) transcription analysis was conducted based on transcriptomic data combined with terpenoid metabolic profiling under different abiotic stress conditions. Totally 80 TPS-like genes were identified. Twenty-three CsTPS genes possessed a complete coding sequence and most likely were functional. The remaining 57 in the currently available database lack essential gene structure or full-length transcripts. Distinct tempo-spatial expression patterns of CsTPS genes were found in tea plants. 17 genes were substantially expressed in all the tested organs with a few exceptions. The other 17 were predominantly expressed in leaves whereas additional eight were primarily expressed in flowers. Under the treatments of cold acclimation, salt and polyethylene glycol, CsTPS67, -69 and -71 were all suppressed and the inhibited expression of many others were found in multiple stress treatments. However, methyl jasmonate resulted in the enhanced expression of the majority of CsTPS genes. These transcription data were largely validated using qPCR. Moreover, volatile terpenoid profiling with leaves, flowers and stress-treated plants revealed a general association between the abundances of mono- and sesqui-terpenoids and some CsTPS genes. These results provide vital information for future studies on CsTPS regulation of terpenoid biosynthesis.

Influence of media supplements on inhibition of oxidative browning and bacterial endophytes of Camellia sinensis var. sinensis.

Explant oxidative browning and necrosis of Camellia sinensis var. sinensis is a severe problem in tissue culture, often associated with the exuded phenolic compounds and microbial contamination from the explants. In this study, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase (PAL), and different antibiotics were tested to control tea explant necrosis and browning. These compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron at different concentrations. Our data indicated that application of 2 µM of AIP was able to effectively inhibit callus browning, significantly reduce EGC abundance, and greatly improve callus induction and growth. Moreover, the use of 150 mg/L of timentin and 30 mg/L gentamycin resulted in an effective elimination of the surface and endophytic microbes associated with explants of C. sinensis var. sinensis. Our study revealed that the inhibition of PAL using AIP combined with the two tested antibiotics could open up new doors to control oxidative tissue browning and endophyte contamination in tissue culture for tea genetic manipulation.

Protocol: high-efficiency in-planta Agrobacterium-mediated transgenic hairy root induction of Camellia sinensis var. sinensis.

BACKGROUND: Camellia sinensis var. sinensis is widely grown for tea beverages that possess significant health promoting effects. Studies on tea plant genetics and breeding are hindered due to its recalcitrance to Agrobacterium-mediated genetic transformation. Among the possible reasons, oxidation of phenolics released from explant tissues and bactericidal effects of tea polyphenols during the process of transformation play a role in the plant recalcitrance. The aim of the present study was to alleviate the harmful effects of phenolic compounds using in-planta transformation. RESULTS: Two-month old seedlings of tea cultivar "Nong Kangzao" were infected at the hypocotyl with wild type Agrobacterium rhizogenes and maintained in an environment of high humidity. 88.3% of infected plants developed hairy roots at the wounded site after 2 months of infection. Our data indicated that transgenic hairy root induction of tea can be achieved using A. rhizogenes following the optimized protocol. CONCLUSION: With this method, composite tea plants containing wild-type shoots with transgenic roots can be generated for "in root" gene functional characterization and root-shoot interaction studies. Moreover, this method can be applied to improve the root system of composite tea plants for a better resistance to abiotic and biotic stresses.