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CsGOGAT is important in dynamic changes of theanine content in postharvest tea plant leaves under different temperature and shading spreadings Zhi-Wei Liu, Hui Li, Wen-Li Wang, Zhi-Jun Wu, Xin Cui, and Jing Zhuang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04552 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Journal of Agricultural and Food Chemistry

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CsGOGAT is important in dynamic changes of theanine

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content in post-harvest tea plant leaves under different

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temperature and shading spreadings

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† † † † † *,† Zhi-Wei Liu , Hui Li , Wen-Li Wang , Zhi-Jun Wu , Xin Cui , Jing Zhuang

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†

Tea Science Research Institute, College of Horticulture, Nanjing Agricultural

University, Nanjing, 210095, People’s Republic of China

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* Corresponding author

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Tel.:+ 86 25 8439 5182; Fax: +86 25 8439 5182

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E-mail address: [email protected] (J.Z.)

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ACS Paragon Plus Environment


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ABSTRACT: We analyzed the changes of theanine content in post-harvest tea

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leaves under high temperature (38 °C), low temperature (4 °C), and shading

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spreadings by using the ultra-high-performance liquid chromatography. The

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differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT,

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which are involved in theanine biosynthesis pathway, were identified from the

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corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and

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CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology.

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The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT

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and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain

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reaction. The correlations between the dynamic changes of theanine content and

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expression profiles of related genes and DEPs were analyzed. This study preliminarily

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proved the the importance of CsGOGAT in dynamic changes of theanine content in

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post-harvest tea leaves during spreading.

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KEYWORDS: Post-harvest leaves; Spreading; Differentially expressed protein;

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Gene expression; CsGOGAT protein; Protein-protein interaction; Tea plant

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■ INTRODUCTION

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Teas are mainly classified into six categories based on how they were processed,

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these categories are green tea (non-fermented), white tea (slightly fermented), yellow

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tea (lightly fermented), oolong tea (semi-fermented), dark tea (post fermented), and

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black tea (fully fermented).1 Spreading is currently an indispensable procedure used to

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improve tea quality and is now widely used in pre-processing of green tea. An

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appropriate spreading condition prior to fixing is beneficial for green tea quality.2

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During spreading, physical and chemical changes of characteristic components in

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post-harvest tea leaves are accompanied by water evaporation.3, 4 The total amount of

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free amino acid shows upward trend, but different amino acids display varying

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trends.5 Studies have shown that L-theanine content decreases after tea shoots

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spreading for 9 h and 18 h.6 Additionally, theanine content increases within a certain

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period and peaks when the moisture content of spread tea leaves is approximately

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70%.2, 7 The maximum theanine level is higher when the post-harvest tea leaves are

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exposed to direct light.7 Studies have demonstrated the dynamic changes of theanine

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content in tea leaves spread process are related with length of spread time, conditions

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and methods.

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Many key enzymes play indispensable role in theanine biosynthesis. Glutamine

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synthetase/glutamate synthase (GS/GOGAT) cycle is the major pathway for

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ammonium assimilation, which produces the precursor glutamate for theanine

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production.8 Two isoforms of GOGAT with different structures, properties and

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functions have been found in plants, these isoforms are ferredoxin-dependent GOGAT



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(Fd-GOGAT ) and nicotinamide adenine dinucleotide-dependent GOGAT

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(NADH-GOGAT).9 Fd-GOGAT assimilates ammonium derived from

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photorespiration in leaves, whereas NADH-GOGAT accumulates in non-green tissues

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and plays important role in ammonium assimilation in roots.10 The mRNA levels,

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enzyme polypeptide and activity of Fd-GOGAT in leaf cells induced by specific

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intensity and quality of light, whereas NADH-GOGAT activity remains low under

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light exposure.11 The tissue expression specificity of Fd-GOGAT and NADH-GOGAT

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gene families were verified in rice seedlings.12 Furthermore, the enzyme gene

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transcript and protein levels, which are influenced by tissue specificity and

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environment conditions, affect the amino acid metabolism.9

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In tea plant growth and development, the appropriate shading is beneficial to

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improve the nutritional and sensory quality of green tea. 13-15 Under the shading

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treatment, the light intensity is changed as well as the temperature than control. The

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stimulation changes of secondary metabolites in tea leaves are also considered as the

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collective effect of light radiation and temperature under shading. During spreading of

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post-harvest tea leaves, ambient temperature and light intensity influence the

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concentration of functional components by affecting the rate of moisture evaporation,

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and the progress and extent of chemical reaction.3, 7, 16-18 In practical process of tea,

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some hours maybe needed to store or transport fresh tea leaves picked from tea plants

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to processing factory. The post-harvest tea leaves are spread in the storage for keeping

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fresh and as well as improving the quality of finished tea. In this study, the 4 °C,

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25 °C and 38 °C were set for tea leaves treatments as the temperature conditions of


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storage.

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In our previous studies, 17 genes of key enzymes involved in theanine metabolism

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pathway were identified from tea plant transcriptome.19 The proteomes of post-harvest

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tea leaves under different spreading treatments, i.e., high temperature (38 °C), low

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temperature (4 °C), room temperature (25 °C) and shading were investigated by

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isobaric tags for relative and absolute quantification (iTRAQ) technology. To explore

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the influence of temperature and light on the dynamic changes of theanine content in

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spreading at the molecular level, the differentially expressed proteins (DEPs) in the

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theanine metabolism pathway were identified at 38 °C, 4 °C and shading treatment

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spreadings in this study. The protein-protein interaction of DEPs and other enzymes

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involved in theanine synthesis were proved, the correlation of DEPs and related genes

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expression levels and theanine content were analyzed. The importance of DEPs was

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firstly verified in dynamic changes of theanine content in post-harvest tea leaves

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under different temperature and shading spreadings.

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■ MATERIALS AND METHODS

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Plant materials and spreading treatments. Two-year-old cuttings of tea plants

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were cultivated in an artificial climate chamber in Nanjing Agricultural University.

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The artificial weather was maintained at 25 °C for 14 h with 150 µmol·m−2·s−1 light

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intensity at daytime and at 18 °C for 10 h during night time. Relative humidity was

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controlled at 70 %. The plant materials were grown in plastic plugs containing peat,

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vermiculite and perlite mixture (volume ratio = 3:2:1). Every one hundred tea cuttings



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seedlings growing well and similar were used for each treatment. On 27 September 2016, tender tea shoots containing 1st, 2nd, 3rd leaves, were

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taken from Camellia sinensis cv. ‘Longjing 43’ tea plants for spreading under shading

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and different temperature treatments (Figure 1). The shading and different

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temperature treatments were performed in light incubator. Under the shading

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treatment, the tea leaves were covered with tin foil paper, and the shading rate was up

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to 100 %. The 25 °C was set for shading treatment for 1 h (S1) and 4 h (S4). Under

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the different temperature treatments, the light density without shading was set as 150

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µmol·m−2·s−1. The 4 °C and 38 °C were set as low and high temperature treatments

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for 4 h. The sample under 25 °C without shading treatment was set as control for 0 h

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(CK0), 1 h (CK2) and 4 h (CK1).

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The post-harvest tea leaves subjected to temperature and shading treatments after

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being spread for 4 h were used in RNA isolation and theanine content determination.

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Three independent biological replicates for each sample were prepared, and the

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samples were immediately frozen in liquid nitrogen and stored at -80 °C.

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Cloning genes of DEPs in theanine biosynthesis pathway. Based on the tea

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transcriptome database,20 the genes of DEPs in theanine biosynthesis pathway were

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identified. These genes were cloned using reverse transcription polymerase chain

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reaction (RT-PCR) with cDNA from tea plant cultivar ‘Longjing 43’ as template. The

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amplification system contained LA-Taq mix 25 µL, cDNA 2.5 µL, forward primer 2.5

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µL, reverse primer 2.5 µL, and ddH2O was added up to 50 µL. The PCR procedure

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was as follows: 95 oC for 5 min; 95 °C 30 s, 55 °C 30 s, 72 °C 5/7 min for 35 cycles


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(with an extension of 5 min for CsFd-GOGAT, 7 min for CsNADH-GOGAT); 72 °C

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for 10 min. The PCR products were detected using 1 % agarose gel electrophoresis,

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and then sequenced by General Biosystems Co., Ltd (Chuzhou, China). The clone

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primer sequences of the genes were listed in Table 1.

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Identification and analysis of DEPs in the theanine biosynthesis pathway.

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Based on the corresponding available iTRAQ-based quantitative proteomics data,21

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the DEPs in the theanine biosynthesis pathway were identified from different samples

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subjected to spreading treatments. BLAST comparison and conserved domain

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prediction of the cloned nucleotide and amino acid sequences of DEPs were

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performed in NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide

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sequences of DEPs from tea plant and other species were compared using DNAMAN

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6.0. Using the Swiss-Model online software (https://swissmodel.expasy.org/), we

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established three-dimensional structural models of DEPs, and edited to build the

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graphics using the Swiss-Pdb Viewer 3.7 software.22 Protein-protein interaction (PPI)

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networks were constructed using the STRING10.5 database

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(http://www.string-db.org/) to better understand the functions and interactions of

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DEPs and theanine biosynthesis related enzymes.23

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Yeast two-hybrid assay. For the yeast two-hybrid assay, the cDNA fragment

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encoding central domain of CsFd-GOGAT was cloned and connected with the

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pGADT7 vector digested by Eco RΙ and Bam HΙ enzymes, the cDNA fragments

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encoding CsTS1 (theanine synthetase), CsNiR (nitrite reductase), and two central

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domains of CsNDAH-GOGAT (aCsNDAH-GOGAT, bCsNDAH-GOGAT) were



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cloned and connected with the pGBKT7 vector digested by Eco RΙ and Sal Ι enzymes.

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Unique amplified primers for cloning these five cDNA fragments were represented in

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Table 1. These vectors connected with the target gene fragments were co-transformed

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into the yeast AH109 strain. All the constructed vectors were co-transformed with

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non-digested pGADT7 or pGBKT7 vector into yeast to verify whether they have

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self-activation. Yeast co-transformation of pGBKT7-53 and pGADT7-T, and the

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co-transformation of pGBKT7-Lam and pGADT7-T were used as positive and

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negative interaction control, respectively.

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Ultra-high performance liquid chromatography (UPLC) determination of

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theanine concentrations. The post-harvest tea leaves subjected to different spreading

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treatments were powdered in liquid nitrogen, and dried until constant weight in a

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freeze dryer. Powered dried tea leaves samples were leached by pure water at 80 °C

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for 45 min (1 g/200 mL H2O) and then centrifuged. The leaching solution was filtered,

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and then mixed the supernatant for theanine content detection. A method based on

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UPLC was developed for the determination of theanine content. A Waters ACQUITY

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UPLC H-class system (Waters, Milford, USA) with a BEH C18 column (2.1×50

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mm,1.7 µm) and a fluorescence (FLR) detector was used for chromatographic

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separation and detection. The volume ratio of mobile phase A (20 mmol·L−1

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ammonium acetate), B (methanol), and C (acetonitrile) was 60 %: 20 %: 20 %. The

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other chromatographic conditions were as follows: flow rate, 0.3 mL/min; detection

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wavelength, 338 nm; column temperature, 40 °C; and injection volume, 2 µL. Prior to

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the injection, the derivative reagent O-phthalaldehyde (OPA) was used for derivative


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reaction with the samples for 2 min. RNA isolation and cDNA reverse transcription. Total RNA was isolated from

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tea leaves by an RNA extraction kit (Aidlab, Beijing, China) according to the

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manufacturer’s instruction. RNA concentration and quality were assessed using a

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Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and

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the agarose gel electrophoresis (10 g·L−1). cDNA was synthesized from total RNA by

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using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China), and then stored at

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–20 °C for quantitative real-time PCR (qRT-PCR) analysis.

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qRT-PCR analysis. To analyze the transcript levels of 17 genes involved in

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theanine metabolic pathways in post-harvest spreading tea leaves under different

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conditions, we searched the sequences of these genes from a transcriptome database

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of tea plant.20 qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa, Dalian,

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China) according to the manufacturer’s instruction for Bio-Rad IQ5 real-time PCR

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System (Bio-Rad, Hercules, CA, USA). Each reaction system contained 10 µL of

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SYBR Premix Ex Taq,7.2 µL of double-distilled water, 2 µL of diluted cDNA, and

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0.4 µL of forward/reverse primer. The reaction conditions were as follows: 95 °C for

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30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s; and 61cycles at 65 °C for 10 s. The

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primer sequences of those related genes of theanine metabolic pathways used for

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qRT-PCR according to Liu et al. 19

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Statistics and analysis. Standard solution samples (0.005, 0.01, 0.02, 0.03, and

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0.05 mg·L−1) of theanine were prepared and used to create a standard curve

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(Supplementary Figure 1). Supplementary Figure 2 shows the UPLC profiles of



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theanine standard solution samples. Gene expression level in non-spreading tea leaves

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sample was defined as 1. The mean values and standard deviation (SD1) for theanine

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contents and gene expression levels were calculated using three independent

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biological replicates. The gene expression levels were calculated relative to the

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CsTBP (TATA-box binding protein) gene by using the 2−∆∆CT method.24 Using the

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SPSS 17.0 software, significant differences in theanine contents and gene expression

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levels were detected by Duncan’s multiple-range test at 5 % significance level, and

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the correlation between the theanine contents and expression levels of genes and

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DEPs were analyzed with Pearson method.25 The column chart was performed with

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Origin 6.0 software.

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■RESULTS Identification of DEPs in theanine biosynthesis pathway in post-harvest

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spreading tea leaves under different treatments. Numerous shoots (1st, 2nd, and

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3rd leaves) of C. sinensis cv. ‘Longjing 43’ were harvested and spread under shading

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for 1 h (S1), 4 h (S4) and under high (38 °C) and low (4 °C) temperature treatments

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for 4 h. The tea leaves spread under normal temperature (25 °C) and not subjected to

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shading treatment for 0, 1, 4 h were marked as CK0, CK2 and CK1, respectively. The

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tea leaves curled, and their color deepened gradually with extended spreading time.

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Obvious changes in the appearance were observed in tea leaves under heat spreading,

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whereas slight change in tea leaves under the shading spreading (Figure 1). The

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number of DEPs in the various compared groups 4 °C/CK1, 38 °C/CK1, S1/CK2,


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S4/CK1 were 566, 635, 537, 857, respectively. The ID and fold change of all DEPs

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are listed in Supplementary Table 2. Only one enzyme CsGOGAT as DEP involved in

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theanine biosynthesis pathway was retrieved from the proteomics data. The

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schematics of theanine biosynthesis pathway in tea plant is shown in Figure 2.

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When tea leaves were spread under cold condition, only CsFd-GOGAT, an

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up-regulated DEP in theanine biosynthesis pathway (Log2(Fold Change) = 1.20) was

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identified. Under heat condition, two DEPs were identified, namely, the up-regulated

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(Log2(Fold Change) = 1.10) CsFd-GOGAT and the down-regulated (Log2(Fold

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Change) = 0.85) CsNADH-GOGAT (Figure 3). CsFd-GOGAT was the only

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up-regulated protein observed in spreading tea leaves both under high and low

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temperatures, and the degree of up-regulation in these conditions was very close.

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In S1 and S4, CsNADH-GOGAT was down-regulated (Log2(Fold Change) = –0.60)

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and CsFd-GOGAT was up-regulated (Log2(Fold Change) = 0.81) (Figure 3).

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CsNADH-GOGAT and CsFd-GOGAT possibly played different roles in tea leaves at

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different times when tea leaves were spread under shading.

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Cloning and analysis of DEPs genes CsFd-GOGAT and CsNADH-GOGAT. The

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DEP genes CsFd-GOGAT and CsNADH-GOGAT were cloned, and the nucleotide and

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amino acid sequences of these genes are listed in Supplementary Figures 3, 4. The

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respective length of the open reading frame (ORF) of CsFd-GOGAT and

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CsNADH-GOGAT were 4881 and 6606 bp, and they correspondingly encode 1627

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and 2202 amino acids. The conserved domains of CsFd-GOGAT and

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CsNADH-GOGAT in tea plant were predicted by NCBI online program. The



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fragment of 2425-3939 bp was the central domain of CsFd-GOGAT, the fragments of

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2500-3972 bp and 5086-6543 bp were the central domain of CsNADH-GOGAT.

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The amino acid sequences of 27 Fd-GOGATs and 21 NADH-GOGATs from

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different species were aligned, and the aligned identities were 88.08 % and 88.19 %,

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respectively (Supplementary Table 3; Supplementary Figure 5). Domains prediction

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and alignment showed that, CsFd-GOGAT and CsNADH-GOGAT were highly

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homologous with the Fd-GOGAT and NADH-GOGAT of other species. Based on the

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templates of Fd-GOGAT from Synechocystis sp. PCC 6803 (1ofd.1.A)26 and

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NADPH-GOGAT from Escherichia coli BL21 (DE3) (2vdc.1.A),27 the

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three-dimensional protein structure of CsFd-GOGAT and CsNADH-GOGAT were

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established, respectively. The identity between the CsFd-GOGAT and 1ofd.1.A was

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59.80 %, and that between CsNADH-GOGAT and 2vdc.1.A was 45.01 %. A total of

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54 α-helix and 49 β-strands were found in CsFd-GOGAT protein, and 57 α-helix and

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44 β-strands were found in CsNADH-GOGAT protein (Supplementary Figure 6).

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Change of theanine contents in post-harvest spreading tea leaves under

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different temperature and shading treatments. To investigate the influence of

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temperature and shading treatments on theanine accumulation, we measured the

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theanine contents in spreading tea leaves under low temperature, high temperature,

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and shading treatments by using UPLC. Figure 4-A, B shows the UPLC profiles of

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theanine in tea leaves under different spreading conditions. Theanine was clearly

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chromatographically separated, and the stable peak of theanine appeared at 3.9 min.

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The heights of the peak were positively correlated with the area of the peaks.


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Supplementary Table 4 shows the detailed initial UPLC data. After spreading for 4 h, the theanine contents of post-harvest tea leaves decreased

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significantly. The theanine content (374.98 mg/100 g) in spreading tea leaves under

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25 °C was significant lower than that in non-spreading sample (459.96 mg/100 g).

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However, there was a significant increase from spreading 1 h to 4 h (Figure 4-D).

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Under 4 °C and 38 °C treatments, the theanine contents in spreading tea leaves were

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significant lower than that under 25 °C. Moreover, the theanine content in cold

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spreading was significant higher than in heat spreading, and a successive significant

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decrease among the 25 °C, 4 °C and 38 °C spreading tea leaves (Figure 4-C). Under

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shading treatment, the theanine content was slightly higher in S4 than in S1, but the

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theanine contents in both treatments were significantly lower than that in CK0. By

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contrast, the theanine content in the control samples significantly decreased in S1 and

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then significantly increased in S4 (Figure 4-D).

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Protein-protein interaction analysis. A string network analysis was performed to

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reveal the protein-protein interaction network of 11 theanine metabolism related

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enzymes (Figure 5). The Arabidopsis thaliana was specified to search for our

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sequences. GS1 (glutamine synthetase), Fd-GOGAT and NADH-GOGAT interacted

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with all the 10 other enzymes. GS2 only interacted without ALT (alanine

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transaminase), the GDHs (glutamate dehydrogenase) only interacted without NR

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(nitrate reductase). The GGT1 and GGT3 (γ-glutamyl transpeptide) interacted with

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five theanine biosynthesis related enzymes, namely, Fd-GOGAT, NADH-GOGAT,

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GS1, GS2, GDH1, and GDH2. However, no direct interaction was observed between



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the GGT1 and GGT3. By contrast, the more interaction evidences was presented

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between the enzymes belonging to same family, such as between Fd-GOGAT and

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NADH-GOGAT, between GS1 and GS2, between GDH1 and GDH2. Moreover, the

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strongest evidence of interaction was observed between Fd-GOGAT and

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NADH-GOGAT.

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To verify the interactions among the proteins, the fusion plasmids of pGADT7 and

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pGBKT7 were co-transformed into the yeast strain AH109. Figure 6 showed that

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co-expression of the CsFd-GOGAT with aCsNADH-GOGAT, bCsNADH-GOGAT,

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CsTS1, or CsNiR resulted in formation of yeast colony in quadruple dropout

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medium (QDO: SD2/-Ade/-His/-Leu/-Trp). The positive control showed that

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pGBKT7-53 can interact with pGADT7-T, but the negative control and the

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self-activation verification did not display co-expression. These results demonstrated

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that CsFd-GOGAT interacted directly with aCsNADH-GOGAT,

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bCsNADH-GOGAT, CsTS1, or CsNiR in yeast.

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Gene expression profile analysis. To validate the DEPs expressions, we

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determined the expression profiles of 17 genes involved in the theanine metabolic

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pathway by using qRT-PCR (Figures 7, 8). The gene expression profiles revealed

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obvious different characteristics among in post-harvest tea leaves under different

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spreading treatments.

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Spreading under different temperature treatments. Generally, the gene expression

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levels were considerably higher in 4 °C spreading than in 38 °C spreading except for

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CsALT. The higher expression levels of most theanine metabolic genes correspond to


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the higher theanine content when tea leaves were spread at 4 °C than that at 38 °C.

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Compared with the gene expression levels in 25 °C spreading, those of all theanine

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metabolic genes were higher in 4 °C spreading except the CsGDH1 and were lower in

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38 °C spreading except the CsALT and CsNADH-GOGAT. Genes belonging to the

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same family, namely, CsTS1 and CsTS2, CsGS1 and CsGS2, CsGGT1 and CsGGT3,

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and CsSAMDC (s-adenosylmethionine decarboxylase) and CsADC (arginine

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decarboxylase), showed consistent expression trend in non-spreading and three

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temperature treatments for 4 h spreadings. However, CsGDH1 and CsGDH2,

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CsFd-GOGAT and CsNADH-GOGAT, and CsCuAO (copper methylamine oxidase)

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and CsPAO (primary amine oxidase), showed different expression profiles (Figure 7).

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Spreading under shading treatment. In tea leaves spread under non-shading

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treatment, the expression levels of CsTS1, CsTS2, CsGS1, CsGDH1, CsGDH2,

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CsALT, CsFd-GOGAT and CsNiR genes significantly decreased at 1 h and then

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increased at 4 h. However, the CsNADH-GOGAT, CsGGT1, CsGGT3 and CsCuAO

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genes exactly showed the opposite expression profiles. The expression levels of

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CsSAMDC, CsADC, CsPAO and CsNR genes decreased gradually in shading

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spreading sample and their expression levels were significantly lower than those in

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control.

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In the shading spreading samples, the expression levels of CsTS1 and CsTS2

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showed no significant change relative to the control. The expression levels of CsNiR

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and CsGS2 gradually increased and decreased, respectively. The expression levels of

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the 13 other genes significantly decreased at 1 h and then increased at 4 h. The



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expression profiles of CsTS1, CsGDH1, CsGDH2, CsALT and CsFd-GOGAT in

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non-shading and shading spreading samples showed the same trend, whereas those of

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CsTS2, CsNADH-GOGAT, CsGGT1, CsGGT3 and CsCuAO showed the opposite

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trend (Figure 8).

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Correlation analysis of theanine content and expression levels of related genes

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and DEPs. The correlation coefficient of theanine content and expression levels of

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genes and DEPs were calculated by Pearson analysis (Figure 9). The correlations were

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analyzed from three aspects, the control, temperature, and shading treatment

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spreading. Among the genes, 13, 13, and 10 genes expression levels were positively

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correlated with the theanine content in control, temperature, and shading spreading.

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The gene expression levels of CsFd-GOGAT was positively correlated with the

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theanine content, whereas the CsNADH-GOGAT was negatively. The protein level of

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CsFd-GOGAT was negatively correlated with the theanine content except under the

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shading treatment, the CsNADH-GOGAT was positively correlated with the theanine

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content.

337 338 339

■ DISCUSSION DEPs in theanine biosynthesis pathway in post-harvest tea leaves under

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different temperatures and shading spreadings. Based on the comparative analysis

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of the available quantitative proteomics data for tea leaves spread under different

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conditions, i.e., 4 °C/CK1, 38 °C/CK1, S1/CK2 and S4/CK1, only GOGATs were the

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identified as DEPs in theanine metabolism pathway. No differential expression of


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other key enzymes involved in theanine metabolic pathway was found. On on hand,

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we speculated that the GOGATs were the only DEPs identified possibly related with

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their large molecular weight and special structure and thus they were detected more

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easily.27, 28 On the other hand, the central position of GOGATs in theanine

348

metabolism pathway, which belongs to ammonium assimilation and nitrogen transport,

349

was indirectly proved in tea plant.9 As shown in protein-protein interaction prediction,

350

the interactions of CsGOGAT with other enzymes involved in theanine synthesis were

351

predicted. Furthermore, the interactions of CsFd-GOGAT with theanine synthase and

352

nitrite synthase were verified by yeast two-hybrid, which proved the important role of

353

CsGOGAT in theanine synthesis.

354

In this study, CsFd-GOGAT only showed up-regulated expression profile, whereas

355

CsNADH-GOGAT only down-regulated among different spreadings. The

356

up-regulated CsFd-GOGAT expression is possibly related to their location in the

357

photosynthetic organ and high levels of mRNA, enzyme polypeptide and activity in

358

leaves induced with light and metabolites.9, 11 Based on STRING database, the

359

identified DEP CsFd-GOGAT in this study displayed high similarity with the

360

AtFd-GOGAT encoded by AtFd-GOGAT1, which is highly expressed primarily in

361

leaves.29

362

Moreover, CsNADH-GOGAT was down-regulated in tea leaves under 38 °C and

363

S1 spreadings containing lower theanine content. NADH-GOGAT is highly

364

accumulated in non-green tissue, and the linear correlation between NADH-GOGAT

365

T-DNA accumulation and glutamate reduction was proved in Arabidopsis root.10 We



366

speculated that CsFd-GOGAT and CsNADH-GOGAT perform distinct and

367

non-overlapping functions in theanine metabolism, and the down-regulated

368

CsNADH-GOGAT expression limits the theanine accumulation.30

369

L-theanine content changes in post-harvest tea leaves under different

370

temperatures spreadings. The theanine contents were significantly lower in all

371

spread tea leaves under different treatments than those that were not spread. This

372

result was obtained on one hand because in vitro tea leaves no longer accept theanine

373

from the root, which as an important organ for theanine synthesis in tea plants.31 On

374

the other hand, the theanine hydrolysis was continuous in tea leaves after harvest, and

375

the degree of hydrolysis was much larger than that of synthesis. Furthermore, the rate

376

of theanine hydrolysis increased with the activity of theanine hydrolase (ThYD)

377

increased in tea leaves harvested within 10 h and then declined gradually.32

378

In tea leaves spread under heat or cold conditions, temperatures quite affected the

379

dynamic changes of theanine. Especially under the high temperature treatment, the

380

activity and gene expression levels of the theanine metabolism related enzymes were

381

strongly suppressed. With the rapid increase in the rate of water evaporation, nitrogen

382

compounds were concentrated continuously and the possibility of exposure to

383

hydrolase was enhanced.2 Moreover, the protein hydrolases were possibly activated

384

and enzymes were probably hydrolyzed with increase of temperature.33

385

DEPs expression profiles in post-harvest tea leaves under different

386

temperatures spreadings. Under temperature treatment spreadings, the level of

387

CsFd-GOGAT was negatively correlated with theanine content, while


Page 18 of 45

Page 19 of 45


388

CsNADH-GOGAT was positively. In shading spreadings, both levels of

389

CsFd-GOGAT and CsNADH-GOGAT were positively correlated with theanine

390

content. In tea plant, the theanine metabolism was not equivalent to the ammonium

391

assimilation. Additionally, the theanine metabolism was the unique pathway in tea

392

plant, which probably caused the different role of CsGOGAT played in tea plant.

393

CsFd-GOGAT expression was up-regulated and not inhibited in spreading tea

394

leaves both under heat and cold treatments. CsNADH-GOGAT was not differentially

395

expressed in cold spreading but was down-regulated in heat spreading. The influence

396

of temperature, particularly high temperature, on CsNADH-GOGAT expression was

397

larger than that on CsFd-GOGAT expression. In 38 °C spreading, the

398

CsNADH-GOGAT positively correlated with theanine content showed

399

down-regulated expression, and the CsFd-GOGAT negatively correlated with

400

theanine content showed down-regulated expression. It may be the reason of lower

401

theanine content in 38 °C spreading than in 4 °C spreading at protein level. It also

402

showed that CsFd-GOGAT cannot compensate for the reduced activity of

403

CsNADH-GOGAT although the CsFd-GOGAT expression level was increased.34

404

Gene expression profiles in post-harvest tea leaves under different

405

temperatures spreadings. The expression levels of 16 genes in theanine metabolism

406

pathway, were significantly higher in cold spreading than heat spreading. Among the

407

16 genes, the expression levels of 13 genes were positively correlated with the

408

theanine content under temperature treatments spreadings. It probably was the cause

409

of that the theanine content in cold spreading was much higher than in heat spreading.



410

The CsFd-GOGAT showed positive correlation with theanine content, while

411

CsNADH-GOGAT showed negative. However, the correlation between theanine

412

content and expression levels of genes were opposite with the proteins levels of DEPs.

413

It revealed that the protein expression was the integrate result of multi-step regulation,

414

thus there is no linearly association from the genetic to the phenotypic level.35

415

Under heat spreading, gene expression levels were strongly suppressed, and the

416

levels were even close to 0. It indicated that high temperature exerted a stronger

417

negative effect on gene expression and theanine accumulation than low temperature.

418

In addition, the gene expression levels in cold spreading were even higher than in

419

normal spreading containing higher theanine content. It revealed that temperature is

420

not the sole factor affecting gene expression,36 and that the expression levels of

421

related genes are not the only factor affecting theanine accumulation.37

422

L-theanine dynamic changes in post-harvest tea leaves under shading

423

spreading. Theanine content in tea leaves under control spreading decreased at 1 h,

424

and then significantly increased at 4 h. It was consistent with the results of Yin et al.

425

(2009) and Too et al. (2015).2, 7 Theanine content initially declined probably because

426

transportation of theanine from the root was cut off suddenly. Tea leaf is the main

427

organ of theanine hydrolysis, and the 25 °C is a suitable hydrolysis temperature, thus

428

both of these factors are not conducive for theanine accumulation. Meanwhile, the

429

glutamic acid used for theanine synthesis is reduced with the decreased activity of

430

glutaminase (GLS).32

431

Under shading treatment, theanine content in spreading tea leaves remained


Page 20 of 45

Page 21 of 45


432

relatively stable after significant decrease. It may be that light and the thermal effect

433

of light promote spreading by accelerating proteolysis and generation of free amino

434

acids.38 It also showed that a reasonable combination of indoor and solar spreading

435

enhances theanine content and fresh taste of tea.

436

DEPs expression profiles in post-harvest tea leaves under shading spreadings.

437

In shading spreading, the DEPs both CsFd-GOGAT and CsNADH-GOGAT levels

438

were positively correlated with the theanine content, and the correlation coefficients

439

were very close. However, the level of CsFd-GOGAT was negatively correlated with

440

theanine content in control spreading, while CsNADH-GOGAT was positively. It

441

evidenced that the CsFd-GOGAT expression was more easily influenced by light than

442

CsNADH-GOGAT.11 The different expression profiles of CsGOGAT may

443

contributed to the different dynamic change of theanine content.

444

Therefore, we speculated that the light affected change of theanine content in vitro

445

tea leaves by regulating the CsGOGAT expression. However, the proteolysis of two

446

chloroplast proteases was responsible for the accumulation of free amino acids in

447

dark-treated in vivo tea leaves.39 It showed that shading regulate different key

448

enzymes related to amino acid accumulation when the tea leaves from in vivo to in

449

vitro, but lead to similar result of improving tea quality by adjusting amino acid

450

content.

451

Gene expression profiles in post-harvest tea leaves under shading spreading. In

452

control and shading spreading, expression profiles of 13 and 10 theanine biosynthesis

453

related genes were positively correlated with the dynamic changes of theanine content,



454

respectively. The expression level of CsFd-GOGAT and CsNADH-GOGAT

455

respectively showed positive and negative relation with the theanine content, and the

456

relational degree in control was greater than in shading spreading. In addition, the

457

structural genes in the theanine biosynthesis pathway, CsTS1, CsTS2, CsGS1, CsGS2,

458

CsGDH1, CsGDH2, CsALT, CsFd-GOGAT, and CsNiR expressed significantly

459

increased from S1 to S4 in control spreading. Those may contribute to the significant

460

increase of theanine content.

461

Although the increase of theanine content in shading spreading tea leaves was not

462

significant despite the significant increase of 14 genes expression levels, the

463

correlation between gene levels and theanine content was presented under shading

464

treatment. Moreover, the expression levels of the structural genes in the theanine

465

biosynthesis pathway were promoted by shading. It may was why the shading

466

treatment can facilitate the theanine accumulation in tea leaves in vivo from the

467

perspective of gene level.40, 41

468

In summary, different expression levels of CsFd-GOGAT and CsNADH-GOGAT

469

were correlated with theanine accumulation in spreading tea leaves both at mRNA

470

and protein levels. In this study, the protein-protein interactions of CsFd-GOGAT and

471

CsNADH-GOGAT, CsTS1, or CsNiR were firstly verified by yeast two-hybrid

472

technology. The importance of CsGOGAT in theanine metabolism in post-harvest tea

473

leaves under different temperature and shading spreadings was evident initially.

474

However, the further functional importance of CsGOGAT and other related enzymes

475

in theanine metabolism pathway will be further verified.


Page 22 of 45

Page 23 of 45


476 477

■ABBREVIATIONS

478

ADC: Arginine decarboxylase

479

AIDA: L-alanine decarboxylase

480

ALT: alanine transaminase

481

AO: amine oxidase

482

CuAO: copper methylamine oxidase

483

DDO: double dropout medium

484

DEP: differentially expression protein

485

DW: dry weight

486

GDH: glutamate dehydrogenase

487

GGT: γ-glutamyl transpeptide

488

GOGAT: glutamine-2-oxoglutarate aminotransferase/glutamate synthase

489

Fd-GOGAT: ferredoxin-dependent glutamate synthase

490

NADH-GOGAT: nicotinamide adenine dinucleotide-glutamate synthase

491

GS: glutamine synthase

492

NiR: nitrite reductase

493

NR: nitrate reductase

494

PAO: primary amine oxidase

495

PPI: protein-protein interaction

496

QDO: quadruple dropout medium

497

qRT-PCR: quantitative real-time PCR



498

SAMDC: S-adenosylmethionine decarboxylase

499

SD1: standard deviation

500

SD2: synthetic dropout medium

501

TS: L-theanine synthetase

502

TBP: TATA-box binding protein

503

UPLC: ultra-high performance liquid chromatography

504 505

■AUTHOR INFORMATION

506

Corresponding Author

507

*Telephone: +86-25-8439-5182; Fax: +86-25-8439-5182.

508

E-mail: [email protected] (J.Z.)

509

Funding

510

The research was supported by the National Natural Science Foundation of China

511

(31570691).

512

Notes

513

The authors declare no competing financial interest.

514


Page 24 of 45

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515

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of shade treatment on theanine biosynthesis in Camellia sinensis seedlings. Plant

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647

■Legends to Figures

648 649

Figure 1 The spreading post-harvest tea leaves under different treatments

650

CK0: no spreading post-harvest tea leaves;

651

CK2: 25oC spreading post-harvest tea leaves for 1 h without shading;

652

CK1: 25oC spreading post-harvest tea leaves for 4 h without shading;

653

S1: 25oC spreading post-harvest tea leaves for 1 h under shading;

654

S4: 25oC spreading post-harvest tea leaves for 4 h under shading;

655

38oC: 38oC spreading post-harvest tea leaves for 4 h without shading;

656

4oC: 4oC spreading post-harvest tea leaves for 4 h without shading

657

Each grid scale in the frame represents 1 cm.

658 659

Figure 2 The schematics of the theanine biosynthesis pathway in tea plant

660

GS: glutamine synthase; GOGAT: glutamine-2-oxoglutarate

661

aminotransferase/glutamate synthase; GDH: glutamate dehydrogenase; ALT: alanine

662

transaminase; AIDA: L-alanine decarboxylase; NiR: nitrite reductase; NR: nitrate

663

reductase. The GOGAT as the only identified DEP in the theanine biosynthesis

664

pathway was marked as yellow.

665 666

Figure 3 The expression profiles of DEPs in the theanine metabolism pathway in

667

spreading tea leaves under different treatments

668



669

Figure 4 The L-theanine ultra-high performance liquid chromatography (UPLC)

670

profiles and L-theanine contents in different spreading tea leaves samples

671

A. L-theanine UPLC profiles of spreading tea leaves under different temperature

672

treatments;

673

B. L-theanine UPLC profiles of spreading tea leaves under shading treatment;

674

C. L-theanine contents in spreading tea leaves under different temperature treatments;

675

D. L-theanine contents in spreading tea leaves under shading treatment

676

Data of L-theanine contents are presented as mean ± SD1 of three independent

677

replicates; Different lowercase letters in parts C and D indicate significant differences

678

at P < 0.05 based on three biologic repetitions, the letters without parentheses in part

679

D present significance of CK samples and the letters in parentheses present

680

significance of shading samples.

681 682

Figure 5 The string network analyses of protein-protein interaction among enzymes

683

in theanine metabolism pathway

684 685

Figure 6 Interaction between CsFd-GOGAT and

686

aCsNADH-GOGAT/bCsNADH-GOGAT/CsTS1/CsNiR in yeast cells. A:

687

CsFd-GOGAT + aCsNADH-GOGAT; B: CsFd-GOGAT + bCsNADH-GOGAT; C:

688

CsFd-GOGAT + CsTS1; D: CsFd-GOGAT + CsNiR. AD: pGADT7-T +

689

aCsNADH-GOGAT/bCsNADH-GOGAT/CsTS1/CsNiR; BD: pGBKT7-T +

690

aCsFd-GOGAT; +: pGADT7 + pGBKT7-53; -: pGADT7 + pGBKT7-lam.


Page 32 of 45

Page 33 of 45


691

Transformants grown on DDO and QDO medium. DDO: SD2/-Leu/-Trp; QDO:

692

SD2/-Ade/-His/-Leu/-Trp.

693 694

Figure 7 The gene expression profiles in spreading tea leaves under different

695

temperature treatments

696

The gene expression level in CK0 was defined as 1.

697

Error bars indicate standard deviation among three independent replicates.

698

Data are presented as mean ± SD1 of three independent replicates.

699

Different lowercase letters indicate significant differences at P < 0.05 based on three

700

biologic repetitions.

701 702

Figure 8 The gene expression profiles in spreading tea leaves under shading treatment

703

The gene expression level in non-spreading sample was defined as 1.

704

Error bars indicate standard deviation among three independent replicates.

705

Data are presented as mean ± SD1 of three independent replicates.

706

Different lowercase letters indicate significant differences at P < 0.05 based on three

707

biologic repetitions, the letters without parentheses present significance of CK

708

samples and the letters in parentheses present significance of shading samples.

709 710

Figure 9 The correlation analyses between theanine content and expression levels of

711

related genes (A) and DEPs (B). The genes from left to right in abscissa of A were

712

followed by CsTS1, CsTS2, CsGS1, CsGS2, CsGDH1, CsGDH2, CsSAMDC, CsADC,



713

CsALT, CsFd-GOGAT, CsNADH-GOGAT, CsGGT1, CsGGT3, CsCuAO, CsPAO,

714

CsNR, and CsNiR.

715


Page 34 of 45

Page 35 of 45


716

Table 1 Sequences of primers used in clone and yeast two-hybrid

717

Gene name

Purpose

forward primer 5'-3'

reverse primer 5'-3'

CsFd-GOGAT

For clone

ATGTCTGTGCAGTCAGTGCCT

TGCAGACTGCAAAGTCACCGT

CsNADH-GOGAT

For clone

ATGCGTGTCTTAGGCCATAAT

CGTCATTACTGTTTGTTTGTT

CsFd-GOGAT

For yeast two-hybrid

GCCATGGAGGCCAGTGAATTCATGGTTACAATTGAGCAAGCTCAA

CAGCTCGAGCTCGATGGATCCCTATACATCTCGTGGTCCTAGTAGATC

aCsNADH-GOGAT


ATGGCCATGGAGGCCGAATTCATGTCAAAAGATGAGCTTGTCAAAAAG

ATGCGGCCGCTGCAGGTCGACCTATTCGAGCATATCTGATCGACCAAC

bCsNADH-GOGAT


ATGGCCATGGAGGCCGAATTCATGCATCTCGAGTTCCAGATGCTATAA

ATGCGGCCGCTGCAGGTCGACCTATTCCCTCATGAGAAACTTGTCAAC

CsTS1


ATGGCCATGGAGGCCGAATTCATGTCTTTGCTATCAGATCTC

ATGCGGCCGCTGCAGGTCGACCTATGGCTTCCACAGCAGAGTGGT

CsGS1


ATGGCCATGGAGGCCGAATTCATGTCTCTTCTTTCCGATCTT

ATGCGGCCGCTGCAGGTCGACCTACGGTTTCCAGAGGATGGTGGT

CsNiR


ATGGCCATGGAGGCCGAATTCATGTCATCTTTTTCAATTAGG

ATGCGGCCGCTGCAGGTCGACCTAATCTTCCCTCTCCTTGAGGAC

718

Tips: The restriction sites were marked with red.

719



Figure 1 The spreading post-harvest tea leaves under different treatments CK0: no spreading post-harvest tea leaves; CK2: 25oC spreading post-harvest tea leaves for 1 h without shading; CK1: 25oC spreading post-harvest tea leaves for 4 h without shading; S1: 25oC spreading post-harvest tea leaves for 1 h under shading; S4: 25oC spreading post-harvest tea leaves for 4 h under shading; 38oC: 38oC spreading post-harvest tea leaves for 4 h without shading; 4oC: 4oC spreading post-harvest tea leaves for 4 h without shading Each grid scale in the frame represents 1 cm. 49x19mm (300 x 300 DPI)


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Figure 2 The schematics of the theanine biosynthesis pathway in tea plant GS: glutamine synthase; GOGAT: glutamine-2-oxoglutarate aminotransferase/glutamate synthase; GDH: glutamate dehydrogenase; ALT: alanine transaminase; AIDA: L-alanine decarboxylase; NiR: nitrite reductase; NR: nitrate reductase. The GOGAT as the only identified DEP in the theanine biosynthesis pathway was marked as yellow. 29x11mm (300 x 300 DPI)



Figure 3 The expression profiles of DEPs in the theanine metabolism pathway in spreading tea leaves under different treatments 44x33mm (300 x 300 DPI)


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Figure 4 The L-theanine ultra-high performance liquid chromatography (UPLC) profiles and L-theanine contents in different spreading tea leaves samples A. L-theanine UPLC profiles of spreading tea leaves under different temperature treatments; B. L-theanine UPLC profiles of spreading tea leaves under shading treatment; C. L-theanine contents in spreading tea leaves under different temperature treatments; D. L-theanine contents in spreading tea leaves under shading treatment Data of L-theanine contents are presented as mean ± SD1 of three independent replicates; Different lowercase letters in parts C and D indicate significant differences at P < 0.05 based on three biologic repetitions, the letters without parentheses in part D present significance of CK samples and the letters in parentheses present significance of shading samples. 129x80mm (300 x 300 DPI)



Figure 5 The string network analyses of protein-protein interaction among enzymes in theanine metabolism pathway 51x37mm (300 x 300 DPI)


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Figure 6 Interaction between CsFd-GOGAT and aCsNADH-GOGAT/bCsNADH-GOGAT/CsTS1/CsNiR in yeast cells. A: CsFd-GOGAT + aCsNADH-GOGAT; B: CsFd-GOGAT + bCsNADH-GOGAT; C: CsFd-GOGAT + CsTS1; D: CsFd-GOGAT + CsNiR. AD: pGADT7-T + aCsNADH-GOGAT/bCsNADH-GOGAT/CsTS1/CsNiR; BD: pGBKT7T + aCsFd-GOGAT; +: pGADT7 + pGBKT7-53; -: pGADT7 + pGBKT7-lam. Transformants grown on DDO and QDO medium. DDO: SD2/-Leu/-Trp; QDO: SD2/-Ade/-His/-Leu/-Trp. 54x20mm (300 x 300 DPI)



Figure 7 The gene expression profiles in spreading tea leaves under different temperature treatments The gene expression level in CK0 was defined as 1. Error bars indicate standard deviation among three independent replicates. Data are presented as mean ± SD1 of three independent replicates. Different lowercase letters indicate significant differences at P < 0.05 based on three biologic repetitions. 170x120mm (300 x 300 DPI)


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Figure 8 The gene expression profiles in spreading tea leaves under shading treatment The gene expression level in non-spreading sample was defined as 1. Error bars indicate standard deviation among three independent replicates. Data are presented as mean ± SD1 of three independent replicates. Different lowercase letters indicate significant differences at P < 0.05 based on three biologic repetitions, the letters without parentheses present significance of CK samples and the letters in parentheses present significance of shading samples. 194x152mm (300 x 300 DPI)



Figure 9 The correlation analyses between theanine content and expression levels of related genes (A) and DEPs (B). The genes from left to right in abscissa of A were followed by CsTS1, CsTS2, CsGS1, CsGS2, CsGDH1, CsGDH2, CsSAMDC, CsADC, CsALT, CsFd-GOGAT, CsNADH-GOGAT, CsGGT1, CsGGT3, CsCuAO, CsPAO, CsNR, and CsNiR. 59x25mm (300 x 300 DPI)


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The graphic for table of contents 47x26mm (300 x 300 DPI)


CsGOGAT Is Important in Dynamic Changes of ... - ACS Publications

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