Transcriptomic and Metabolic Insights into the Distinctive Effects of

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Transcriptomic and metabolic insights into the distinctive effects of exogenous melatonin and gibberellin on terpenoid synthesis and plant hormone signal transduction pathway in Camellia sinensis. Taimei Di, Lei Zhao, Huimin Chen, Wenjun Qian, Peiqiang Wang, XinFu Zhang, and Tao Xia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00503 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of Agricultural and Food Chemistry

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Transcriptomic and metabolic insights into the distinctive effects of

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exogenous melatonin and gibberellin on terpenoid synthesis and

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plant hormone signal transduction pathway in Camellia sinensis.

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Taimei Di1, Lei Zhao1, Huimin Chen1, Wenjun Qian1, Peiqiang Wang1, Xinfu

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Zhang1*, Tao Xia2*.

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1

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2

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University, Hefei 230036, China;

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*Authors to whom correspondence should be addressed, E-mails: [email protected]

College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China; State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

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(Xinfu Zhang); [email protected] (Tao Xia)

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Tel: +86-0532-86080740

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Fax: +86-0532-86080740

ACS Paragon Plus Environment


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ABSTRACT:

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Melatonin and gibberellin are bioactive molecules in plant. In present study, the role

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of exogenous melatonin (MT) and gibberellin (GA) on tea plant was explored by

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transcriptome and metabolic analysis. Results showed that the growth of tea plant was

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enhanced by MT treatment. The pathways of terpenoid synthesis and plant-pathogen

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interaction were significantly strengthened, combined with the up-regulation of

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LRR-RLK and transcription factors contributed to the growth of tea plant. The

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internode elongation and leaf enlargement were hastened by GA treatment.

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Significantly modulated expression was occurred in the plant hormonal signal

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transduction, complemented by the up-regulation of phenylpropanoid biosynthesis

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and expansins to achieve growth acceleration, while, the flavonoid synthesis was

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repressed in GA treatment. Therefore, the distinctive effect of MT and GA treatment

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on tea plant was different. The MT exhibited significant promotion in terpenoid

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synthesis, especially, TPS14 and TPS1. GA was prominent in coordinated regulating

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plant hormonal signal transduction.

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Keywords: melatonin, gibberellin, Camellia sinensis, terpenoid synthesis, plant

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hormonal signal transduction


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INTRODUCTION

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Tea plant (Camellia sinensis (L.) O. Kuntze), an important perennial evergreen

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leaf-used economic crop, distributes across the tropical and subtropical regions of the

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earth1. Tea young shoot with rich metabolites confers on tea unique flavors and

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healthy function2, 3. Plant hormones is a group of small molecules with diverse and

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effective physiological activity and involves in the regulation of plant growth and

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development as a chemical messengers 4.

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MT was discovered in the bovine pineal gland in mammalian5 and was identified

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in plants in 19956, 7. MT took part in regulating plant growth and development.

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Hussain M et.al reported MT enhanced photosynthesis in tomato8. Back K et.al

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studied MT promoted seed germination9. The study of Zhao B et.al indicated MT

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promoted root architecture10 and delayed flowering11. Zheng G et.al studied MT

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delayed leaf senescence12 and Zhang N et.al reported MT promoted fruit ripening13.

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MT acted as a significant role in increasing the plant biotic and abiotic stress

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resistance and performs an efficient free radical scavenger role for hydroxyl radical

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(.OH), superoxide anion (O2.-) and hydrogen peroxide (H2O2) in plant systems14. The

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MT also exhibited protective effect on Camellia sinensis L. under cold stress15, 16.

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Gibberellins (GAs), tetracyclic diterpenoids, are indispensable stimulators in plants

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growth and development. GAs is involved in various processes in plants. such as

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regulating shoot elongation in Arabidopsis17, promoting seed germination in

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Arabidopsis18, fruit enlargement in pear19, inhibiting senescence in alstroemeria

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leaves20 and so on. Yuerong Liang reported that application of GAs was beneficial to

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green tea quality based on the chemical composition21. Chuan Yue studied the

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regulation role of GA in the bud activity-dormancy transition of tea plants22.

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Exogenous MT exhibited excellent function on other species, while, the study on



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tea plant was little and the regulatory effect of MT on tea plant growth and quality

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formation remains unclear. In protected tea plantation, exogenous GAs is often used

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to accelerate tea plant growth during young shoot germination period. However, the

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effects of exogenous GA on the appearance and nutritional quality of tea remain

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largely unknown. Therefore, further study was needed to understand the role of

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exogenous MT and GA in tea plant growth and development, then, providing

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guidance for agriculture production. In the present study, we investigated the

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transcriptome and metabolic responses of tea plant to exogenous MT and GA aiming

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to explore the regulation of MT and GA on tea plant growth and tea quality formation.

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MATERRIALS AND METHOD

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Plant materials, growth conditions and treatments

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One-year old tea plant Camellia sinensis (L.) O. Kuntze cv. ‘Zhongcha 108’ as

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plant materials were used in this study. HalfHoagland nutrient solution was used as

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hydroponic growth medium and the growth conditions were maintained at 25/20◦C

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(day/night) air temperature with 80% relative humidity, photoperiod of 12 h light /12

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h darkness. After acclimating for one week, tea young shoot and leaves was sprayed

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with MT (100 μM) and GA (100 μM) under non light every two days, which was

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according to Li, X15 and Li, J16. Equal-volume deionized water was used as the

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control. Plants were grown in the greenhouse in May at Qingdao, Shandong Province,

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China. The solutions were refreshed every two days. The tea plant was sprayed three

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times according to the above description, at two hours after the third times treatment,

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tea young shoot with one bud and two leaves were gently rinse and wipe dry, then

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collected as samples from three individuals with three biological replicates, and

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immediately frozen in liquid nitrogen, and stored at -80◦C.

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RNA extraction, library construction and RNA-seq data processing and analysis


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Total RNA was extracted with Trizol reagent (Invitrogen, USA), and DNA

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contaminants were removed by RNase-Free DNase I (TaKaRa, Japan). Agarose gel

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electrophoresis and an Agilent 2100 Bioanalyzer (Agilent, USA) confirmed the RNA

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integrity. RNA concentrations were measured by a Qubit 2.0 spectrophotometer (Life

84

Technology, USA). According to the manufacturer’s instructions (Invitrogen, USA),

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mRNA of each treatment was purified and fragmented by magnetic oligo (dT) beads

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and fragmentation buffer with the preparation kit. The first-strand cDNA was

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synthesized by the cleafd short RNA fragments using reverse transcriptase and

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hexamer primer (Illumina), and the DNA polymerase I and RNase H (Termo Fisher,

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USA) were used for second strand cDNA synthesis. PCR amplification using cDNA

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fragments, and cDNA libraries were used to sequence on the IlluminaHiSeq™ 2000

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platform23.

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The clean reads were de novo assembled into unique consensus sequence using 24

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Trinity

and mapped to the reference sequence of tea plant using tophat

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(version2.0.9)

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mismatches and multihits ≤ 1 and the final transcriptome assembled from the resulting

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assemblies using CD-HIT-EST v4.626. Differentially expressed genes (DEGs) were

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defined with a threshold of |log2FC| > 1 and the P vlaue < 0.05. The identified DEGs

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were subjected to enrichment analysis of Gene Ontology (GO) functions and KEGG

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pathway analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and

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Genomes (KEGG) enrichment analysis was carried out using the OmicShare tools

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(www.omicshare.com/ tools). The raw transcriptome sequencing data are available on

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NCBI SRA (Sequence Read Archive, http://www.ncbi.nlm.nih.gov/sra/) repository

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under accession number PRJNA516040.

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Metabolite Profiling Analysis

(http://www.plantkingdomgdb.com/tea_tree/)25,


allowed

two


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The 100 mg freeze-dried samples powder was extracted overnight at 4 °C with

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1.0 ml 70% aqueous methanol (containing 0.1 mg/L lidocaine), centrifugation at 4 °C,

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10,000 g for 10 min, then absorbing and filtering the supernatant. Metabolite profiling

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analysis was performed using an LC-ESI-MS/MS system (UPLC, Shim-pack

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UFLCSHIMADZU CBM20A, http://www.shimadzu.com.cn/; MS/MS (Applied

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Biosystems 4500 QTRAP, http://www.appliedbiosystems.com.cn/). Three biological

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replicates for each treatment (CK, MT, GA) were independently performed and

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analysed. Five μl of samples were injected onto a Waters ACQUITY UPLC HSS T3

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C18 column (2.1 mm*100 mm, 1.8 µm) operating a flow rate of 0.4 mL/min and at

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40°C, Phase A was acidified water (0.04 % acetic acid) and Phase B was acidified

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acetonitrile (0.04 % acetic acid). The following gradient was performed to separate

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the compounds: 95:5 Phase A/Phase B at 0 minutes; 5:95 Phase A/Phase B at 11.0

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minutes; 5:95 Phase A/Phase B at 12.0 minutes; 95:5 Phase A/Phase B at 12.1

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minutes; 95:5 Phase A/Phase B at 15.0 minutes.

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The effluent from the above column was connected to an ESI triple

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quadrupole-linear ion trap (Q TRAP)–MS. LIT and triple quadrupole (QQQ) scans

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were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP)

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equipped with an ESI-Turbo Ion-Spray interface under AB Sciex QTRAP4500

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System, a positive ion mode and controlled by Analyst 1.6.1 software (AB Sciex).

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The following parameters were operated: ESI source temperature 550°C; ion spray

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voltage (IS) 5500 V; the collision-activated dissociation (CAD) was set to the highest

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setting; curtain gas (CUR) 25psi. QQQ scans were acquired as MRM experiments

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with optimized declustering potential (DP) and collision energy (CE) for each

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individual MRM transitions. The range of m/z was set between 50 and 1000.

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Data pre-processing and metabolites identification were performed by Analyst


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1.6.1 software, searching internal database and public databases, as well as comparing

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the m/z values, the RT, and the fragmentation patterns with the standards. The

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variable importance of the projection (VIP) score of the application (O) PLS model

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was used to filter the best differentiated metabolites between treatments. Metabolites

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with a P value of T test of 0.7 or < -0.7,

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P-value < 0.05) (Figure. 8), which showed positive relationship of the above genes.

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Reinforcement of plant hormone signal transduction, phenylpropanoid

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biosynthesis and expansins under exogenous GA treatment

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KEGG analysis revealed significant enrichment in pathway of plant hormone

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signal transduction, phenylpropanoid biosynthesis and flavonoid biosynthesis in CK

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vs GA. The vital genes in phenylpropanoid biosynthesis were significantly activated

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by

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(coniferyl-alcohol glucosyltransferase), CAD (cinnamyl-alcohol dehydrogenase),

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HCT

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dehydrogenase), F5'H (ferulate-5-hydroxylase), 4CL (4-coumarate-CoA ligase),

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COMT (caffeic acid 3-O-methyltransferase), CSE (caffeoylshikimate esterase) and

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C3'H (5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase).

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flavonoid biosynthesis were significantly repressed by GA, such as F3'H (naringenin

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3-dioxygenase), CHI (chalcone isomerase), LAR (leucoanthocyanidin reductase), ANS

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(anthocyanidin synthase), ANR (anthocyanidin reductase), FLS (flavonol synthase),

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F3'5'H (flavonoid 3',5'-hydroxylase), CCOAMT (caffeoyl-CoA O-methyltransferase),

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DRF

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2''-O-beta-L-rhamnosyltransferase) (Figure.6-B-b). Additionally, the expansin genes

GA

(Figure.6-B-a),

(shikimate

(flavanone

including

β-GLU

(beta-glucosidase),

O-hydroxycinnamoyltransferase),

4-reductase)

and

C12RT1


REF1

UGT72E

(coniferyl-aldehyde

While, key genes in

(flavanone

7-O-glucoside

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(CSA020689, CSA023438, CSA016873, CSA009350, CSA009709, CSA031028,

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CSA017997, CSA017998, CSA017621, CSA030980) were promoted by GA

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(Figure.6-B-c). The genes related to GAs, auxin, cytokinin, BR, ABA and ethylene in

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plant hormone signal transduction pathway were regulated differently by GA in

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different way (Figure.7).

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In GA-signal transduction, three GID1 were down regulated and one DELLA was

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upregulated by GA. In IAA-signal transduction, one and three AUX/IAA was

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significantly up and down regulated by GA. One GH3 and four SAUR were enhanced,

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as well as three ARF were down regulated by GA. The genes involved in cytokinin

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signaling transduction were consistently exhibited down regulation, including CRE1

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(arabidopsis histidine kinase 2/3/4 (cytokinin receptor)) and A-ARR (two-component

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response regulator ARR-A family), as well as AHP (histidine-containing

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phosphotransfer peotein) and B-ARR (two-component response regulator ARR-B

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family). Two BZR1/2 (brassinosteroid resistant 1/2) and one CYCD3 (cyclin D3) in

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brassinosteroid-signal transduction were up regulated and down regulated by GA,

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respectively. In ABA-signal transduction, GA significantly up regulated three PP2C

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(protein phosphatase 2C), four PRY-PYL (abscisic acid receptor PYR/PYL family)

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and one ABF (ABA responsive element binding factor). One gene encoding ERFs

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(ethylene-responsive transcription factors) was found significantly enhanced after GA

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application.

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downregulated by GA.

One

EIN2

(ethylene-insensitive

protein


2)

was

significantly


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To validate the accuracy and repeatability of the transcriptome analysis,

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qRT-PCR was performed on a set of DEGs selected randomly from the above genes.

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Results showed that the expression profiles of the twelve DEGs as determined by

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qRT-PCR were basically consistent with their abundance changes identified by

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RNA-seq (Figure.9), which verified the reproducibility and credibility of RNA-seq

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data. Results confirmed that G6PD, otsB, GERD, TPS14, LUT1, CDPK, RPS2 and

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RPM1 were up-regulated by exogenous MT, and the CH3, SAUR, pp2C and 4CL were

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up-regulated by exogenous GA.

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Metabolomic analysis

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A total of 31 metabolites differentially expressed were obtained. The number of

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metabolites up regulated and down regulated in CK vs GA, CK vs MT, MT vs GA

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were 18 and 12, 8 and 7, 7 and 8, respectively (Figure. 10-A). We could overall

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observe the metabolites pattern from hierarchical clustering analysis (Figure. 10-B).

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The green bands indicated low metabolites quantity, and the red represented the high

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metabolites quantity. The high quantity of L-phenylalanine, phenylpyruvic acid and

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hormone (methoxyindoleacetic acid and 5-methoxy-3-indoleaceate) and the low

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quantity of flavonoids (myricitrin, naringenin chalcone and naringenin), flavone

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(tricetin

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(resokaempferol 7-O-hexoside and pinobanksin) and mannitol 1-phosphate validated

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the transcriptome changes in GA treatment. In CK vs MT, high quantity of trehalose,

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MT, N-methyltyramine, guanosine, crotonoside, LysoPC 18:1 (2n isomer), and low

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quantity of 10-Formyl-THF and 2, 6-diaminooimelic acid were accumulated in MT

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treatment. These changes in metabolite levels under exogenous MT and GA treated

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are approximately consistent with the gene expression changes in the transcriptome.

O-hexoside

and

apigenin

7-O-glucoside


(Cosmosiin)),

flavonol

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Thereby, it can be concluded that the increase and reduction in the contents of

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metabolites under exogenous MT and GA treated are a consequence of regulation of

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mRNA expression patterns.

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DISCUSSION

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The regulation of exogenous MT and GA on tea plant was investigated in our

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study. Omics data analyses revealed that MT, as a multi regulatory molecule,

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participated in promoting terpenoid synthesis and plant-pathogen interaction of tea

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plant; exogenous GA induced internode elongation and leaves enlargement includes a

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balance and reinforcement of plant hormone signal transduction, as well as up

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regulating phenylpropanoid biosynthesis and expansins expression. The action of two

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hormones was different and distinctive.

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The role of MT in stress resistance enhancement, growth and development

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improvement have been fully verified in previous studies as an efficient free radical

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scavenger for hydroxyl radical in plant systems14, 30. While, there is still no report

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about the action of MT on terpenoid synthesis. Terpenoids take part in tea beverage

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aroma formation and the quality and productivity of tea plantation31. However, the

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regulation of terpenoid synthetase remains unclear so far. Some reports have indicated

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terpenoid synthesis varied over growing seasons, and was affected by abiotic stresses,

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biotic stresses, insect attacks32 as well as jasmonate treatment33. In our present study,

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exogenous MT exhibited positive effect on the genes involved in terpenoid synthesis,

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including TPS1 for terpineol, TPS14 for linalool, ASF1 for farnesene, cyc2 and GERD

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for gennacrene, CYP71D55 for solavetivone, LUP2 and LUP4 for amyrin. Terpenoid

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volatiles were regarded as chemical messengers in tea plant and key odorants of tea

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with high popularity34, 35. Linalool, geraniol and α-terpineol were the vital terpenoid

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compounds for tea aromatic flavor36,

37.

The up-regulation of genes related to



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terpenoid synthesis indicates the potential role of MT in tea aromatic aroma

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formation, which provides a young thought to the regulation of terpenoids synthesis.

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The resistance enhancement of tea plant under exogenous MT treatment was reflected

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in

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serine/threonine-protein kinase, transcription factor, as well as the synthesis of

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trehalose. Trehalose, an important carbohydrate, preserves plant from various stresses

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through enhancing their cellular integrity38. Transcription factors (TFs) play important

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roles in regulating the expression of genes involved in terpenoid biosynthesis39, 40 and

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stress resistance in tea plant41,

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related to terpenoid biosynthesis, plant-pathogen interaction, LRR receptor-like

357

serine/threonine-protein kinase and transcription factor in co-expression analysis

358

indicated that exogenous melatonin exhibited reinforcement on tea plant growth.

strengthen

of

plant-pathogen

42.

interaction,

LRR

receptor-like

The strong positive relationship of those genes

359

Exogenous GA3 exhibited coordinated regulation in key points in plant hormone

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signaling transduction in tea plant. In the auxin-response pathway, the expression of

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SAUR and CH3 were enhanced, which was beneficial for cell elongation and

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expansion43,

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regulates GAs response and three PP2C up-regulated in ABA-signal transduction

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negatively regulates ABA response. Exogenous GA promote the expression of ERFs

365

in ethylene-signal transduction, in addition, ERFs could enhance GA signal

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transcription45. So, the nexuses of GAs and ethylene were strengthened, then, may

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contribute to the growth of tea young shoot and internode elongation. The consistent

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repressed phenomenon in the cytokinin downstream seemed to the in line with the

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reported study that GA could inhibit cytokinin during cell maturation and

370

elongation46. The non-covalent bonds expansion was critical step in plant cell-wall

44.

One DELLA up-regulated in GAs-signal transduction positively


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relaxation47. The significant up-regulation of expansion genes suggested their

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contribution to cell-wall relaxation during the process of leaf enlargement and

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internode elongation. In addition, The inhibition effect of exogenous GA on the

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flavonoid synthesis is consistent with the reports in Scutellaria baicalensis Georgi48

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and grapevine49. Flavonoids are ubiquitous secondary metabolites synthesized by the

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phenylpropanoid pathway. The flavonoid synthesis is one of two major branches in

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phenylpropanoids pathway, the inhibition of flavonoid synthesis and the

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encouragement of phenylpropanoids synthesis revealed the other branch enhancement

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of monolignol/lignin biosynthesis which is responsible for producing a suite of

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metabolites fundamental for cytoskeleton, helping tea plant growth. The growth of tea

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young shoot was accelerated, but the flavonoid synthesis was inhibited in GA

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treatment, which indicated exogenous GA may be not favorable to tea flavor quality.

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Therefore, the balance of yield and quality should be considered in exogenous GA

384

efficacy evaluation.

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Our findings offered a global view and comparison on the effect of exogenous

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MT and GA on tea plant, which aided in the understanding of molecular process that

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tea plant in response to exogenous MT and GA and provide young thoughts for

388

agriculture production. Further study should be carried out for the specific molecular

389

mechanisms of MT and GA on tea plants growth, development and quality formation.

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ABBREVIATIONS USED

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MT, melatonin; GA, gibberellin; DEGs, differentially expressed genes; DEMs,

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differentially expressed metabolites; Nr, NCBI protein database; GO, Gene Ontology;

393

KEGG, Kyoto Gene and Genome Encyclopedia; ultra-high performance liquid



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chromatography coupled to a triple quadrupole mass spectrometer; FDFT1,

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farnesyl-diphosphate farnesyltransferase; AFS1, alpha-farnesene synthase; GERD,

396

(-)-germacrene D synthase; cyc2, (-)-gennacrene D synthase; CYP71D55,

397

premnaspirodiene oxygenase; LUP2, lupeol synthase 2; LUP4, beta-amyrin synthase;

398

TPS14, (3S)-linalool synthase;

399

trimethyltridecatetraene/dimethylnonatriene

400

enzymes;

401

4-coumarate-CoA ligase.

402

CONTRIBUTION

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Xinfu Zhang and Tao Xia conceived and designed the experiments. Taimei Di,

404

Huimin Chen and Peiqiang Wang performed the experiments. Taimei Di, Lei Zhao

405

and Wenjun Qian analyzed the data. Xinfu Zhang and Taimei Di wrote the

406

manuscript. All authors read and approved the manuscript.

407

FUNDING SOURCES

408

This work was supported by Open Fund of State Key Laboratory of Tea Plant Biology

409

and Utilization (SKLTOF20150110), Postgraduate Innovation plan Project of

410

Qingdao Agricultural University (QYC201720) and Talents Start-up funds of

411

Qingdao Agricultural University (663/1114343).

412

CONFLICT OF INTEREST

413

The authors declare no conflict of interest

414

Supporting Information.

415

Table S1. Throughput and quality summary of RNA-sequence. CK group treated with

416

deionized water; MT group treated with 100 μΜ MT.GA group treated with 100 μΜ GA.

TPS1, (-)-alpha-terpineol synthase; CYP82G1, synthase;

GH3,

auxin-conjugating

SAUR, Small Auxin-Up RNA; pp2c, protein phosphatase 2C; 4CL ,


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Table S2. Genes for co-expression analysis. DEGs encoding transcription factor, LRR

418

receptor-like serine/threonine-protein kinase, as well as DEGs involved in terpenoid

419

biosynthesis and plant-pathogen interaction in MT treatment.

420

Table S3. Primer sequence for qRT-PCR.



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REFERENCE

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(1)

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Wu, Z. J.; Li, X. H.; Liu, Z. W.; Xu, Z. S.; Zhuang, J., De novo assembly and transcriptome

characterization: novel insights into catechins biosynthesis in Camellia sinensis. Bmc Plant Biology 2014, 14, 277. DOI: 10.1186/s12870-014-0277-4. (2)

Wang, X. C.; Zhao, Q. Y.; Ma, C. L.; Zhang, Z. H.; Cao, H. L.; Kong, Y. M.; Yue, C.; Hao, X.

Y.; Liang, C.; Ma, J. Q., Global transcriptome profiles ofCamellia sinensisduring cold acclimation. Bmc Genomics 2013, 14, 415. DOI: 10.1186/1471-2164-14-415. (3)

Weng, H.; Zeng, X.; Li, S.; Kwong, J.; Liu, T.; Wang, X., Tea Consumption and Risk of Bladder

Cancer:

A

Dose-Response

Meta-Analysis.

Front

Physiol

2016,

7,

693.

DOI:

10.3389/fphys.2016.00693. (4)

Kucera, B.; Cohn, M. A.; Leubnermetzger, G., Plant hormone interactions during seed dormancy

release and germination. Seed Science Research 2005, 15, 281-307. DOI: 10.1079/SSR2005218. (5)

Lerner, A. B.; Case, J. D.; Takahashi, Y.; Lee, T. H.; Mori, W., Isolation of melatonin, the pineal

gland factor that lightens melanocytes1. Journal of the American Chemical Society 1958, 80, . DOI: 10.1021/ja01543a060. (6)

Hattori, A.; Migitaka, H.; Iigo, M.; Itoh, M.; Yamamoto, K.; Ohtanikaneko, R.; Hara, M.; Suzuki,

T.; Reiter, R. J., Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem Mol Biol Int 1995, 35, 627-634 (7)

Dubbels, R.; Reiter, R. J.; Klenke, E.; Goebel, A.; Schnakenberg, E.; Ehlers, C.; Schiwara, H. W.;

Schloot, W., Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography‐mass spectrometry. Journal of Pineal Research 1995, 18, 28-31. DOI: 10.1111/j.1600-079x.1995.tb00136.x. (8)

M, H.; M, I.; S, M.; M, L.; S, L.; D, Q., Exogenous Melatonin Mitigates Acid Rain Stress to

Tomato Plants through Modulation of Leaf Ultrastructure, Photosynthesis and Antioxidant Potential. Molecules 2018, 23, undefined. DOI: 10.3390/molecules23020388. (9)

HJ, Z.; QQ, S.; YY, C.; X, L.; B, Z.; P, W.; YD, G., Proteomic analysis reveals a role of

melatonin in promoting cucumber seed germination under high salinity by regulating energy production. Scientific reports 2017, 7, 503. DOI: 10.1038/s41598-017-00566-1. (10) K, B., Melatonin promotes seminal root elongation and root growth in transgenic rice after

germination. Journal of pineal research 2012, 53, 385-9. DOI: 10.1111/j.1600-079X.2012.01008.x. (11) K, B., An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including

enhanced seedling growth, delayed flowering, and low grain yield. Journal of pineal research 2014, 56, 408-14. DOI: 10.1111/jpi.12129. (12) G, Z.; W, L.; Y, W.; B, H.; H, W.; H, W.; Y, Q.; XG, Z.; DX, T.; SY, C.; C, C., Melatonin delays

leaf senescence and enhances salt stress tolerance in rice. Journal of pineal research 2015, 59, 91-101. DOI: 10.1111/jpi.12243. (13) N, Z.; J, W.; H, Z.; D, L.; J, S.; R, L.; S, W.; B, Z.; S, R.; YD, G., Melatonin promotes ripening

and improves quality of tomato fruit during postharvest life. Journal of experimental botany 2015, 66, 657-68. DOI: 10.1093/jxb/eru332. (14) S, W.; N, Z.; X, Z.; G, N.; Y, G.; GA, B.; C, F.; S, R., Arabidopsis transcriptome analysis reveals

key roles of melatonin in plant defense systems. PloS one 2014, 9, e93462. DOI: 10.1371/journal.pone.0093462. (15) Li, X.; Wei, J.; Scott, E.; Liu, J.; Guo, S.; Li, Y.; Zhang, L.; Han, W., Exogenous Melatonin


Page 21 of 31

464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507


Alleviates Cold Stress by Promoting Antioxidant Defense and Redox Homeostasis in Camellia sinensis L. Molecules 2018, 23, . DOI: 10.3390/molecules23010165. (16) Li, J.; Arkorful, E.; Cheng, S.; Zhou, Q.; Li, H.; Chen, X.; Sun, K.; Li, X., Alleviation of cold

damage by exogenous application of melatonin in vegetatively propagated tea plant ( Camellia sinensis (L.) O. Kuntze). Scientia Horticulturae 2018, 238, 356–362. DOI: 10.1016/j.scienta.2018.04.068. (17) Sven, E.; Henrik, B. H.; Thomas, M.; Ove, N., GA4 is the active gibberellin in the regulation of

LEAFY transcription and Arabidopsis floral initiation. Plant Cell 2006, 18, 2172-2181. DOI: 10.1105/tpc.106.042317. (18) Ludmila, T.; Thomas, S. G.; Jianhong, H.; Alyssa, D.; Alonso, J. M.; Ecker, J. R.; Tai-Ping, S.,

Della proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiology 2004, 135, 1008-1019. DOI: 10.1104/pp.104.039578. (19) Jaeyoung, L.; Jungmyung, L., Promotion of fruit enlargement of 'Whangkeumbae' Asian pear

fruits by gibberellins. Horticulture Environment & Biotechnology 2006, 47, 183-187 (20) Kappers, I. F.; Jordi, W.; Maas, F. M.; Stoopen, G. M.; Plas, L. H. W. V. D., Gibberellin and

phytochrome control senescence in alstroemeria leaves independently. Physiologia Plantarum 2010, 103, 91-98. DOI: info:doi/10.1034/j.1399-3054.1998.1030111.x. (21) Liang, Y.; Lu, J.; Shang, S., Effect of Gibberellins on Chemical Composition and Quality of Tea

(Camellia sinensisL). Journal of the Science of Food & Agriculture 2015, 72, 411-414. DOI: 10.1002/(sici)1097-0010(199612)72:43.0.co;2-9. (22) Yue, C.; Cao, H.; Hao, X.; Zeng, J.; Qian, W.; Guo, Y.; Ye, N.; Yang, Y.; Wang, X., Differential

expression of gibberellin- and abscisic acid-related genes implies their roles in the bud activity-dormancy

transition

of

tea

plants.

Plant

cell

reports

2018,

37,

425-44110.1007/s00299-017-2238-5. (23) Yu, C.; Zhao, X.; Qi, G.; Bai, Z.; Wang, Y.; Wang, S.; Ma, Y.; Liu, Q.; Hu, R.; Zhou, G.,

Integrated analysis of transcriptome and metabolites reveals an essential role of metabolic flux in starch accumulation under nitrogen starvation in duckweed. Biotechnology for Biofuels 2017, 10, 167. DOI: 10.1186/s13068-017-0851-8. (24) Grabherr, M. G.; Haas, B. J.; Moran, Y.; Levin, J. Z.; Thompson, D. A.; Ido, A.; Xian, A.; Lin, F.;

Raktima, R.; Qiandong, Z., Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 2011, 29, 644. DOI: 10.1038/nbt.1883. (25) Trapnell, C.; Pachter LSalzberg, S. L., TopHat: discovering splice junctions with RNA-Seq.

Bioinformatics 2009, . DOI: 10.1093/bioinformatics/btp120. (26) Fu, L.; Niu, B.; Zhu, Z.; Wu, S.; Li, W., CD-HIT: accelerated for clustering the next-generation

sequencing data. Bioinformatics 2012, 28, 3150-3152. DOI: 10.1093/bioinformatics/bts565. (27) Wang, W.; Zhou, Y.; Wu, Y.; Dai, X.; Liu, Y.; Qian, Y.; Li, M.; Jiang, X.; Wang, Y.; Gao, L.,

Insight into Catechins Metabolic Pathways of Camellia sinensis Based on Genome and Transcriptome Analysis. J Agric Food Chem 2018, 66, acs.jafc.8b00946. DOI: 10.1021/acs.jafc.8b00946. (28) Pfaffl, M. W., A new mathematical model for relative quantification in real-time RT-PCR.

Nucleic Acids Research 2001, . DOI: 10.1093/nar/29.9.e45. (29) Little, C. H.; Macdonald, J. E., Effects of exogenous gibberellin and auxin on shoot elongation

and vegetative bud development in seedlings of Pinus sylvestris and Picea glauca. Tree Physiology 2003, 23, 73-83. DOI: 10.1093/treephys/23.2.73. (30) Arnao, M. B.; Hernández-Ruiz, J., Functions of melatonin in plants: a review. Journal of Pineal

Research 2015, 59, 133-150. DOI: 10.1111/jpi.12253.



508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551

Page 22 of 31

(31) S, Z.; X, W.; X, Y.; L, G.; X, M.; Q, X.; J, Z.; A, W.; L, L.; C, W., Revealing of MicroRNA

Involved Regulatory Gene Networks on Terpenoid Biosynthesis in Camellia sinensis in Different Growing Time Points. Journal of agricultural and food chemistry 2018, 66, 12604-12616. DOI: 10.1021/acs.jafc.8b05345. (32) Fang, D.; Ziyin, Y.; Susanne, B.; Yasushi, S.; Tatsuo, A.; Naoharu, W., Herbivore-induced

volatiles from tea (Camellia sinensis) plants and their involvement in intraplant communication and changes in endogenous nonvolatile metabolites. Journal of Agricultural & Food Chemistry 2011, 59, 13131-5. DOI: 10.1021/jf203396a. (33) J, S.; C, M.; D, Q.; H, L.; T, Y.; Q, P.; Z, C.; Z, L., Transcriptional responses and flavor volatiles

biosynthesis in methyl jasmonate-treated tea leaves. BMC plant biology 2015, 15, 233. DOI: 10.1186/s12870-015-0609-z. (34) Pichersky, E.; Gershenzon, J., The formation and function of plant volatiles: perfumes for

pollinator attraction and defense. Current Opinion in Plant Biology 2002, 5, 237-243. DOI: 10.1016/S1369-5266(02)00251-0. (35) Cho, J. Y.; Shimizu, M. I., Chemical Profiling and Gene Expression Profiling during the

Manufacturing Process of Taiwan Oolong Tea "Oriental Beauty". Bioscience Biotechnology & Biochemistry 2007, 71, 1476-1486. DOI: 10.1271/bbb.60708. (36) Jumtee, K.; Komura, H.; Bamba, T.; Fukusaki, E., Predication of Japanese green tea (Sen-cha)

ranking by volatile profiling using gas chromatography mass spectrometry and multivariate analysis. Journal of Bioscience & Bioengineering 2011, 112, 252-255. DOI: 10.1016/j.jbiosc.2011.05.008. (37) Han, Z. X.; Rana, M. M.; Liu, G. F.; Gao, M. J.; Li, D. X.; Wu, F. G.; Li, X. B.; Wan, X. C.; Wei,

S., Green tea flavour determinants and their changes over manufacturing processes. Food Chemistry 2016, 212, 739-748. DOI: 10.1016/j.foodchem.2016.06.049. (38) Moon, M. H.; Ryu, J.; Choeng, Y. H.; Hong, S. K.; Kang, H. A.; Yong, K. C., Enhancement of

stress tolerance and ethanol production in Saccharomyces cerevisiae by heterologous expression of a trehalose biosynthetic gene from Streptomyces albus. Biotechnology & Bioprocess Engineering 2012, 17, 986-996. DOI: 10.1007/s12257-012-0148-5. (39) Natalia, D.; Antje, K.; Muhlemann, J. L. K.; Ian, K., Biosynthesis, function and metabolic

engineering of plant volatile organic compounds. New Phytologist 2013, 198, 16-32. DOI: 10.1111/nph.12145. (40) Xu, Q.; He, Y.; Yan, X.; Zhao, S.; Zhu, J.; Wei, C., Unraveling a crosstalk regulatory network of

temporal aroma accumulation in tea plant ( Camellia sinensis ) leaves by integration of metabolomics and

transcriptomics.

Environmental

&

Experimental

Botany

2018,

149,

81-94.

DOI:

10.1016/j.envexpbot.2018.02.005. (41) XH, L.; ZW, L.; H, L.; YX, W.; J, Z., Transcriptome-wide identification of Camellia sinensis

WRKY transcription factors in response to temperature stress. Molecular genetics and genomics : MGG 2016, 291, 255-69. DOI: 10.1007/s00438-015-1107-6. (42) Zhao, L.; Gao, L.; Wang, H.; Chen, X.; Wang, Y.; Yang, H.; Wei, C.; Wan, X.; Xia, T., The

R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid biosynthesis. Functional & Integrative Genomics 2013, 13, 75-98. DOI: 10.1007/s10142-012-0301-4. (43) Spartz, A. K.; Sang, H. L.; Wenger, J. P.; Gonzalez, N.; Itoh, H.; Inzé, D.; Peer, W. A.; Murphy,

A. S.; Overvoorde, P. J.; Gray, W. M., The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion. Plant Journal 2012, 70, 978-90. DOI: 10.1111/j.1365-313X.2012.04946.x. (44) Keun, C.; Isaacs, C. G.; Reeves, P. H.; Maloney, G. S.; Muday, G. K.; Punita, N.; Reed, J. W.,


Page 23 of 31

552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568


Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation. Plant Journal 2012, 71, 684-697. DOI: 10.1111/j.1365-313X.2012.05024.x. (45) Hattori, Y.; Nagai, K.; Furukawa, S.; Song, X. J.; Kawano, R.; Sakakibara, H.; Wu, J. Z.;

Matsumoto, T.; Yoshimura, A.; Kitano, H., The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 2009, 460, 1026-30. DOI: 10.1038/nature08258. (46) Weiss, D.; Ori, N., Mechanisms of cross talk between gibberellin and other hormones. Plant

Physiology 2007, 144, 1240-1246. DOI: 10.1021/cm100566v. (47) Moralescruz, A.; Amrine, K. C. H.; Blancoulate, B.; Lawrence, D. P.; Travadon, R.; Rolshausen,

P. E.; Baumgartner, K.; Cantu, D., Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens. BMC Genomics,16,1(2015-06-19) 2015, 16, 469. DOI: 10.1186/s12864-015-1624-z. (48) Y, Y.; Y, L.; C, W.; S, C.; Z, W.; Z, Y.; S, Q.; L, H., Water deficit affected flavonoid

accumulation by regulating hormone metabolism in Scutellaria baicalensis Georgi roots. PloS one 2012, 7, e42946. DOI: 10.1371/journal.pone.0042946. (49) G, M.; A, F.; M, P.; R, B.; G, B.; PN, P., ABA and GA regulate the synthesis of primary and

secondary metabolites related to alleviation from biotic and abiotic stresses in grapevine. Phytochemistry 2017, 135, 34-52. DOI: 10.1016/j.phytochem.2016.12.007.



569

FIGURES CAPTION

570

FIGURE 1. Tea young shoot phenotype under deionized water, MT and GA. CK: control

571

group sprayed with deionized water; MT: MT group sprayed with 100 μM MT; GA: GA

572

group sprayed with 100 μM GA.

573

FIGURE 2. Statistics of the differentially expressed genes (DEGs) between CK and MT, CK

574

and GA, MT and GA. CK: control group sprayed with deionized water; MT: MT group

575

sprayed with 100 μM MT; GA: GA group sprayed with 100 μM GA.

576

FIGURE 3. GO term analysis of the DEGs between CK and MT, CK and GA. CK: control

577

group sprayed with deionized water; MT: MT group sprayed with 100 μM MT; GA: GA

578

group sprayed with 100 μM GA.

579

FIGURE 4. KEGG pathway analysis of the DEGs between CK and MT, CK and GA, MT

580

and GA. CK: control group sprayed with deionized water; MT: MT group sprayed with 100

581

μM MT; GA: GA group sprayed with 100 μM GA.

582

FIGURE 5. The response of genes related to terpenoid biosynthesis and sugar down-stream

583

pathway under exogenous MT and GA treatment. CK: control group sprayed with deionized

584

water; MT: MT group sprayed with 100 μM MT; GA: GA group sprayed with 100 μM GA.

585

FIGURE 6. (A) Fold change of DEGs between MT and CK (Log2 (FC)= Log2 (MT/CK)).

586

A-a: DEGs encoding LRR receptor-like serine/threonine-protein kinase; A-b: DEGs involved

587

in plant-pathogen interaction. (B) Fold change of DEGs between GA and CK (Log2 (FC)=

588

Log2 (GA/CK)). B-a: genes related to phenylpropanoid biosynthesis; B-b: DEGs related to

589

flavonoid synthesis; B-c: DEGs encoding expansion protein. CK: control group sprayed with

590

deionized water; MT: MT group sprayed with 100 μM MT; GA: GA group sprayed with 100

591

μM GA.

592

FIGURE 7. The response in plant hormone signal transduction to exogenous GA treatment.

593

GA: GA group sprayed with 100 μM GA.

594

FIGURE 8. qRT-PCR analysis of a set of DEGs. The expression at CK was set as 1, and the

595

relative expression level was calculated for several genes. G6PD (glucose-6-phosphate

596

1-dehydrogenase), otsB (trehalose 6-phosphate phosphatase), GERD ((-)-germacrene D

597

synthase), TPS14 ((3S)-linalool synthase), LUT1 (carotene epsilon-monooxygenase), CDPK


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598

(calcium-dependent protein kinase), RPS2 (disease resistance protein RPS2), RPM1 (disease

599

resistance protein RPM1), GH3 (auxin-conjugating enzymes), SAUR (Small Auxin-Up RNA),

600

pp2c (protein phosphatase 2C), 4CL (4-coumarate-CoA ligase). CK: control group sprayed

601

with deionized water; MT: MT group sprayed with 100 μM MT; GA: GA group sprayed with

602

100 μM GA.

603

FIGURE 9. Co-expression analysis of genes related to terpenoid biosynthesis, plant-pathogen

604

interaction, LRR receptor-like serine/threonine-protein kinase and transcription factor. Nodes

605

represented gene and edges represented the relationship between any two genes. The purple,

606

yellow, blue and orange nodes represented genes related to terpenoid biosynthesis,

607

plant-pathogen

608

transcription factor, respectively. Blue edge represented positive correlations, determined by a

609

Pearson correlation coefficient > 0.7.

610

FIGURE 10. (A) Statistics of the differentially expressed metabolites (DEMs) (adjusted P
1) between CK and MT, CK and GA, MT and GA. (B)

612

Identification of the all DEMs based on the three samples, CK, MT, GA. The green bands

613

identified low metabolites quantity and the red represented the high metabolites quantity. CK:

614

control group sprayed with deionized water; MT: MT group sprayed with 100 μM MT; GA:

615

GA group sprayed with 100 μM GA.

interaction,

LRR

receptor-like

serine/threonine-protein


kinase

and


Figures Fig.1

Fig.2

Fig.3


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Page 27 of 31


Fig.4

Fig.5



Fig.6

Fig.7


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Fig.8

Fig.9



Fig.10 (A)

(B)


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Transcriptomic and Metabolic Insights into the Distinctive Effects of

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