Transcriptomic and Phytochemical Analyses Reveal Root-Mediated

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Agricultural and Environmental Chemistry

Transcriptomic and phytochemical analysis reveal rootmediated resource-based defense response to leafherbivory by Ectropis oblique in tea plant (Camellia sinensis) Hua Yang, Yanan Wang, Longbao Li, Fangdong Li, Yaxian He, Jianqiang wu, and Chaoling Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00195 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Journal of Agricultural and Food Chemistry

1

Transcriptomic

2

root-mediated

3

leaf-herbivory by Ectropis oblique in tea plant (Camellia

4

sinensis)

and

phytochemical

resource-based

analysis

defense

reveal

response

to

5 6

Hua Yang 1,2*, Yanan Wang 1, Longbao Li 2, Fangdong Li 1,2, Yaxian He 1, Jianqiang

7

Wu 3, Chaoling Wei 1#

8 9

1 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

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University, 130 Changjiang West Road, Hefei, 230036, China

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2 Department of Applied Chemistry, School of Science, Anhui Agricultural

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University, 130 Changjiang West Road, Hefei, 230036, China

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3 Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of

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Botany, Chinese Academy of Sciences, Kunming, 650201, China

15 16

#

17

Email address:

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[email protected] (Hua Yang)

19

[email protected] (Yanan Wang)

20

[email protected] (Longbao Li)

21

[email protected] (Fangdong Li)

22

[email protected] (Yaxian He)

23

[email protected] (Jianqiang Wu)

Corresponding author: [email protected] ; Tel/Fax: +86 551 65786765

1

ACS Paragon Plus Environment


24

[email protected] (Chaoling Wei)

2


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ABSTRACT

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Leaf-herbivory on tea plants (Camellia sinensis) by tea geometrids (Ectropis oblique)

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severely threaten the yield and quality of tea. In previous work, we found that local

28

defense response was induced in damaged leaves by geometrids at transcriptome level.

29

Here, we investigated the systemic response triggered in undamaged roots and the

30

potential role of roots in response to leaf-herbivory. Comparative transcriptome

31

analysis and carbohydrate dynamics indicated that leaf-herbivory activated systemic

32

carbon reallocation to enhance resource investment for local secondary metabolism.

33

The crucial role of JA and the involvement of other potential hormone signals for

34

local and systemic signaling networks were supported by phytohormone

35

quantification and dynamic expression analysis of phytohormone-related genes. This

36

work represents a deep understanding of the interaction of tea plants and geometrids

37

from the perspective of systems biology, and reveals that tea plants have evolved

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intricate root-mediated resource-based resistance strategy to cope with geometrid

39

attack.

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KEYWORDS

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Camellia sinensis; Ectropis oblique; leaf-herbivory; systemic root response; carbon

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reallocation; phytohormone signaling

3



43

INTRODUCTION

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Plants have formed complex and ingenious defense systems in response to insect

45

herbivory during the evolution. Inducible defense response is the resistance generated

46

only when plants are attacked by insect herbivores, which is one of the important

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strategies employed by plants.1 Due to economical utilization of plant resource and

48

efficient exertion of defense roles, inducible defense confers environmental adaptation

49

to plants. Until now, the inducible defense in local response elicited in the damaged

50

leaves by insect herbivory has been detailed deciphered in many plant species.1, 2

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Recently, studies have increasingly found systemic response can also be triggered in

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belowground roots by leaf-herbivory.3 The synthesis of defensive chemicals and

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proteins in roots playing one of the most important roles for enhancement of plant

54

resistance has been documented in tobacco and maize plants.4-7 Another potential role

55

of root is as a mediator of induced tolerance-defense trade-offs through a dynamic

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storage of primary metabolites. The observed increase of carbon allocation to the

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roots in the GAL83-silenced tobacco plants attacked by Manduca sexta,8 and the

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rapid changes in carbohydrate transport and partitioning to storage organs in Populus

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induced by jasmonic acid (JA),9 indicated that the herbivory-induced resource

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sequestration may act as a tolerance mechanism. However, several studies in tobacco

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recently discovered leaf-herbivore attack can also reduce carbon reserves and

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regrowth from the roots, indicating that roots may serve as dynamic producers and

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storage of resources and nutrients that can be transported through vascular and

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utilized for aboveground defense.10, 11 Besides, the active roles of roots in defense

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against leaf attacker involve environmental sensors and root–shoot signal emitters.12,

66

13

4


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The research of local and systemic molecular responses on the transcriptional

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level facilitates us to further comprehend the molecular mechanism of the intricate

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plant-insect interaction and the potential role of the root for the inducible defense. In

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recent years, the transcriptional changes generated in roots in response to

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leaf-herbivory have been investigated in poplar,14 maize,15,

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tobacco.18 These studies discovered that transcriptional changes involved in primary

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and/or secondary metabolism, defense and signal transduction can be induced in

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systemic response, and these changes can be different from those elicited in local

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responses by leaf attackers. However, based on the comparisons of the above

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mentioned studies, leaf-herbivory in different plant species can trigger various and

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complex local and systemic responses.

16

cabbage,17 and

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Insect herbivory elicits complex local and systemic signaling networks to

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mediate local and systemic responses in plants. In the induced signaling networks, JA

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or JA derivatives are usually pivotal phytohormones acting as the essential

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components of local and systemic signals.19-21 In addition, local signaling network has

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been known that it contains various early signaling events, such as recognition of

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elicitors by multiple receptors, initiation of Ca2+ flux and calcium sensors,

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mitogen-activated protein kinases (MAPKs) signaling, production of reactive oxygen

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species (ROS), activation of transcription factors, JA biosynthesis and perception, and

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crosstalk of JA and other phytohormones.1,

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events involved in leaf-to-root systemic signaling mechanism have been poorly

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elucidated until now. Some emerging evidences that crosstalk of JA and other

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phytohormones were also employed in roots in response to leaf-herbivory have been

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recently provided, such as the elicitation of JA and ethylene (ET) signaling in maize,16

91

JA and auxin (IAA) signaling in Nicotiana attenuata,10, 24 and JA and salicylic acid

19, 22, 23

However, the early signaling

5



92

(SA) signaling in pepper plants.25 Current studies suggested that the distinct systemic

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signaling mechanisms were triggered in different plant species or by different kinds of

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insect herbivores. However, the previous research mainly focused on herbaceous

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plants, the data with respect to systemic root response and signaling network in

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woody plants induced by leaf-herbivory were severely insufficient.

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Tea plant is an economically important woody species, whose leaves are

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processed into numerous kinds of tea serving as one of health-promoting beverages.26,

99

27

However, larvae of tea geometrids, one of the most common insect herbivores of

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tea plants in China, can cause seriously chewing damage on leaves, and severely

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threaten the yield and quality of tea.28, 29 Local defense response in damaged tea

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leaves induced by geometrid attack has been detailed studied. Cai et al. provided the

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chemical evidence on emission of volatiles induced by geometrid attack.30 Our and

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Wang’s previous work revealed that local defense response was induced in damaged

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leaves by geometrid attack at transcriptome level.28, 29 Wang et al. further discovered

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geometrid attack induced local defense at transcriptomic and metabolomic level.31

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However, until now, little is known about systemic response elicited in roots by

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leaf-herbivory, the role of roots for the resistance of tea plants, and the systemic

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signaling mechanism underlying the interaction of tea plant with E. oblique.

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In the present study, to investigate the role of roots in tea plants in response to

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leaf-herbivory by geometrids, the local and systemic transcriptome profiling were

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generated and compared to decipher the differences of molecular mechanisms

113

between local and systemic responses. Phytohormone measurement and the analysis

114

of dynamic expressional pattern of phytohormone-related genes permitted us to

115

further comprehend the local and systemic phytohormone signaling. The study

116

represents a valuable resource for deep understanding the important roles of roots and 6


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the responsive systemic phytohormone signaling for the inducible resistance in tea

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plants by leaf-herbivory.

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MATERIALS AND METHODS

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Plant materials and insect infestation

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All plant materials were collected from the two-year-old tea plants (Camellia

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sinensis cv. Shuchazao) growing in plastic pots (35 cm in height, 30 cm in diameter)

123

with consistent water and fertilizer management in the tea plantation of Anhui

124

Agricultural University, Hefei, China. The healthy tea plants with uniform height and

125

canopy width were selected for insect herbivory and mechanical damage treatments.

126

After E. oblique insects at the third larval stage were starved for 8 h, they were

127

distributed evenly on the leaves (20 insects per tea plant) until 1/3 of each leaf was

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consumed. After quick washing the adherent mud off the roots with sterile water, the

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rest of the attacked leaves (EL samples) and the tender lateral roots (ER samples)

130

from the same plants by leaf-herbivory were collected. In the mechanical damage

131

treatment, tea leaves were damaged by autoclaved scissors to an extent similar to E.

132

oblique damage. Samples in three biological replicates were collected at 3, 6, 9, 12,

133

and 24 h after leaf-herbivory or mechanical damage treatment. Leaves and roots from

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tea plants without treatment were used as control samples (control-leaf, CKL;

135

control-root, CKR). All the collected samples were quickly frozen in liquid nitrogen

136

and then stored at -80°C for further use.

137

RNA-Seq, de novo assembly and functional annotation

138

Total RNA was extracted from the collected samples using the RNAprep Pure Plant

139

Kit (Tiangen, China) following the manufacturer’s instruction. Equal amounts of 7



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RNA from samples collected at 3, 6, 9, 12, and 24 h after leaf-herbivory were pooled

141

for EL, ER, CKL and CKR samples, and three biological replicates were obtained for

142

each

143

spectrophotometry (Nanodrop, ThermoFisher, USA) was used for the preparation of

144

sequencing cDNA libraries as described by Tai et al.,32 and the quality of the

145

sequencing libraries was examined using an Agilent 2100 Bioanalyzer. Finally, the

146

libraries were sequenced using Illumina HiSeq™ 2000 at Beijing Genomics Institute

147

(Shenzhen, China). For each sequencing libraries, high-quality clean reads were

148

generated after quality control, and they were mixed and assembled using the Trinity

149

short-read assembling program.33 All assembled unigenes were annotated using

150

BLASTx against the NR, Swiss-Port, and clusters of orthologous groups (COG)

151

databases with an E-value threshold of 1e-5.34 Gene ontology (GO) classification and

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KEGG pathways were also assigned to the assembled unigenes as described by Shi et

153

al.27

154

Identification of differentially expressed genes

155

To identify the genes induced by E. oblique attack, all assembled unigenes from our

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transcriptomes and other published transcriptomes induced by E. oblique attack,28, 29,

157

31

158

genes. Clean reads in each of the twelve RNA-Seq dataset were mapped against the

159

reference genes, and the gene expression level of each gene was quantified according

160

to the amounts of uniquely mapped reads by the method of fragments per kilobase of

161

exon model per million mapped reads (FPKM) using Cufflinks (version 1.0.3).35

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DEGseq R package,36 was applied to identify the differentially expressed genes

163

(DEGs) in EL versus CKL (EL-vs-CKL, referred to as local group) and in ER versus

of

them.

High-quality RNA

validated

by

gel

electrophoresis

and

and the gene models from tea plant genome,26 were combined used as reference

8


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CKR (ER-vs-CKR, referred to as systemic group). To judge the significance of

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differential expression of each gene, the false discovery rate (FDR) ≤ 0.001 and the

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absolute value of log2Ratio ≥ 1 were set as the thresholds.

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Quantitation of phytohormones

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The extraction and quantitation of phytohormones (JA, JA-Ile, ABA, and SA) were

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performed in the leaves and roots of EL, ER, CKL and CKR samples (n = 9) collected

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at 3, 6, 9, 12, and 24 h after leaf-herbivory. Approximately 100 mg fresh weight of

171

sample was ground to powder in liquid nitrogen. The powder, with the addition of a

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mixture of internal standards including 200 ng [2H2] JA and 40 ng each of

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JA-[13C6]-Ile, [2H4] SA and [2H6] ABA, was extracted with 1 ml of ethyl acetate by

174

vortexing for 10 min. After centrifugation at 16,000 g for 15 min at 4 °C, the

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supernatant was obtained and then evaporated until dry using a vacuum concentrator

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(Eppendorf, Germany) at 30 °C. The dried residues were diluted in 500 µL of 70%

177

methanol and then filtered through a 0.22 mm cellulose acetate filter. Phytohormones

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were separated and detected using ProntoSIL C18 analytical column (50 × 2 mm, 5

179

μm) and a 1200 HPLC (Varian, USA) coupled with a triple quadrupole mass

180

spectrometer (API 5000 LC/MS/MS, Applied Biosystems, USA). Phytohormone

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contents were quantified based on the ratio between their ion intensity with their

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respective internal standards.

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GC-MS analysis of carbohydrate metabolites

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The collected tea leaf and root samples (n = 9) were dried by a lyophilizer (-60 °C)

185

and then ground into powder in liquid nitrogen. Each tea sample (200 mg) was

186

extracted three times with methanol/water solution (1:1, v/v). The supernatant was

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pooled and adjusted to 10 ml with a suitable volume of the methanol/water solution. 9



188

For the preparation of the samples for GC-MS analysis according to the protocol of

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Zhang et al.,37 with a few modifications. Briefly, ribitol was added into the extract as

190

an internal standard with the final concentration of 0.1 mg·mL-1. 100 µL of the

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resultant extract was lyophilized under low temperature (-60 °C). The dried sample

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was dissolved in 100 µL of hydroxylamine hydrochloride (25 mg·mL-1 in pyridine)

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and incubated at 70 °C for 50 min for oximation reaction. Subsequently,

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trimethylsilylation reaction was performed at 70 °C for 2 h after the addition of 100

195

µL of BSTFA-TMCS [N, O-Bis-(trimethylsilyl)-trifluoroacetamide containing 1%

196

trimethylchlorosilane] into the sample. The derived sample was equilibrated to room

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temperature before analysis. Authentic standards of D-fructose, D-glucose, sucrose,

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L-malic acid and citrate and samples were respectively analyzed an Agilent HP-5MS

199

column (19091S-436, 30 m × 0.25 mm × 0.25 μm, Agilent Technologies, CA, USA)

200

and an Agilent 7890B GC system (Agilent Technologies, CA, USA) coupled with an

201

Agilent 7000B mass detector Agilent Technologies, CA, USA). Qualitative Analysis

202

of MassHunter Acquisition Data software (version B.07.00) was used for data

203

acquisition.

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qRT-PCR analysis

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The gene-specific primers were designed using Primer Premier software (version 6.0;

206

Supplementary Table S1). Total RNA was extracted using the RNAprep Pure Plant

207

Kit (Tiangen, China), and then was used for the synthesis of single-stranded cDNA

208

using the PrimeScript RT Reagent Kit (Takara). The qRT-PCR reaction was

209

performed containing 300 nM each primer and 2 µL 10-fold diluted cDNA template.

210

The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an

211

internal reference gene. All qRT-PCR analyses were performed in three biological 10


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and three technical replications. The relative expression was calculated using the

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2−ΔΔCt method.38

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RESULTS

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Differential transcriptome reconfiguration between leaf and root

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induced by leaf-herbivory

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To investigate the local and systemic responses on transcriptome level induced in

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damaged leaves and undamaged roots after E. oblique attack on leaves, a total of 12

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sequencing libraries were generated from CKL, EL, CKR and ER samples, with 3

220

biological replicates per sample. The output of high-quality clean reads from

221

paired-end Illumina RNA-Seq added up to 47.5 Gb (Supplementary Table S2). In

222

total, 86,487, 77,781, 92,013 and 90,783 unigenes were constructed based on de novo

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assembly for CKL, EL, CKR and ER samples, respectively (Supplementary Table S3).

224

By combining all non-repetitive unigenes into a whole reference transcriptome dataset,

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102,845 unigenes were achieved with a N50 size of 1,153 bp and average length 724

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bp (Supplementary Table S3). A total of 68,907 (67.0%) unigenes were annotated,

227

containing 63,588 (92.3 %), 41,126 (59.7 %), 37,179 (54.0 %), 23,813 (34.6 %),

228

46,027 (66.8 %) annotations assigned against NR, Swiss-Prot, KEGG, COG and GO

229

databases, respectively (Supplementary Fig. S1~S3 and Table S4). The annotated

230

unigenes were classified into 25 COG categories (Supplementary Fig. S2), 48 GO

231

subgroups in three main groups (Supplementary Fig. S3) and 100 KEGG pathways

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(Supplementary Table S4).

233

Based on the comparative transcriptome analysis, a total of 5,418 DEGs (3,769

234

up-regulated and 1,649 down-regulated genes) from local group, and 9,912 DEGs

235

(7,050 up-regulated and 2,852 down-regulated genes) from systemic group were 11



236

identified, respectively. Furthermore, there were 334 common DEGs found between

237

the two groups, but 5,084 and 9,568 DEGs were specific in local and systemic groups,

238

respectively (Figure 1a). According to the KEGG pathway annotations, more systemic

239

DEGs were involved in seven subcategories of 'metabolism' category (carbohydrate,

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lipid, energy, nucleotide, amino acid and other amino acid metabolism, and

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biosynthesis of secondary metabolites), three subcategories of 'genetic information

242

processing' category (translation; transcription; and folding, sorting and degradation)

243

and one subcategory of 'cellular processes' category (transport and catabolism),

244

whereas more local DEGs were found in four subcategories, including metabolism of

245

terpenoids and polyketides, biosynthesis of other secondary metabolites, signal

246

transduction, and environmental adaptation (Figure 1b). These results suggested that

247

leaf-herbivory triggered a larger scale of transcription reconfiguration occurred in

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undamaged roots than in damaged leaves, and the induced molecular mechanism of

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systemic response was distinct from that of local response.

250

Activation of local defense genes

251

To compare the transcriptional characterization of the local and systemic defense

252

responses, defense genes involved in several secondary metabolism pathways, cell

253

wall remodeling and resistant proteins were investigated. For secondary metabolism, a

254

large amount of genes involved in biosynthesis of phenylpropanoids, flavonoids,

255

caffeine, lignins and terpenoids were activated in local group. In contrast, a slight

256

activation of these genes was found in systemic group. Similarly, PPO (polyphenol

257

oxidase) and MRP (multidrug resistance protein) genes were mainly activated in local

258

group. However, genes encoding cellulose synthases (CSs) and xyloglucan

259

endotransglucosylase/hydrolases (XTHs) responsible for cell wall remodeling and 12


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β-Gluc (resistant proteins β-1,3-glucanase), PI (protease inhibitor) and PR

261

(pathogenesis-related protein) genes were obviously enhanced in systemic group but

262

slightly up-regulated in local group. In addition, chitinase genes and genes associated

263

with biosynthesis of green leaf volatiles (GLV) were actively employed in both local

264

and systemic groups (Figure 2 and Supplementary Table S5). For most of above

265

mentioned defense genes, tissue-specific transcripts were observed between the two

266

groups. Taken together, a great deal of defense genes, especially genes involved in

267

secondary metabolism pathways, were highly activated for the elicitation of local

268

defense response in response to leaf-herbivory on tea leaves.

269

Induction of genes related to depletion of systemic carbon reserves

270

To investigate the balance between the secondary metabolism and the primary

271

metabolism, the DEGs related to primary metabolism pathways were identified in

272

local and systemic groups and further compared each other. We discovered that

273

primary metabolism DEGs were disturbed in both two groups, but leaf-herbivory has

274

a more notable impact on the primary metabolism in systemic group, such as

275

carbohydrate, lipid, amino acid and energy metabolism (Figure 1b). Especially,

276

carbohydrate metabolism was the most heavily affected in systemic group, and the 5

277

top subcategories were involved in the catabolism of carbohydrate, containing starch

278

and sucrose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway,

279

citrate cycle and pyruvate metabolism (Figure 3). It indicated that leaf-herbivory

280

dramatically reduced carbon reserves in systemic response in tea plants, which may

281

be much more reallocated and utilized to produce defensive secondary metabolites in

282

local response.

13



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Different systemic signaling network comparing to local signaling

284

network induced by leaf-herbivory

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The attacked plants firstly perceive insect herbivory, and then trigger elaborate

286

signaling networks, which then play pivotal roles to mediate defense-tolerance

287

trade-offs in plants. To investigate the signaling networks evoked by leaf-herbivory in

288

tea plants, we dissected local and systemic gene expression profilings related to

289

herbivory-induced pattern-recognition receptors (PRRs), Ca2+ signaling, MAPK

290

signaling,

291

transcription factors. For herbivory-induced PRRs, we discovered that 7 out of 22

292

local DEGs related to plasma membrane-localized receptor kinases (RKs) and all 5

293

systemic DEGs for leucine-rich repeat-containing receptor-like kinases (LRR-RLKs)

294

were up-regulated in local and systemic groups, respectively. Especially, 2 LRR-RLK

295

genes were more dramatically enhanced in the systemic group. For Ca2+ signaling,

296

genes encoding Ca2+ sensor proteins, such as calcium-binding protein (CBP),

297

calcium-transporting ATPase (Ca2+-ATPase), calmodulin (CAM), calcineurin B-like

298

protein (CBL), CBL-interacting protein (CBLI) and calcium-dependent protein kinase

299

(CDPK) were activated in local group, especially 4 highly up-regulated CBP and

300

Ca2+-ATPase genes. By comparison, 3 CBP genes and 2 CDPK genes in systemic

301

group were more dramatically enhanced. We found that 11 and 1 up-regulated MAPK

302

genes in local and systemic groups, respectively, suggesting MAPK signaling were

303

employed to exerted stronger effects to mediate the local response in response to tea

304

geometrid attack (Supplementary Figure S4a and Supplementary Table S5). For

305

combating oxidative stress, genes encoding glutathione reductases (GRs), glutathione

306

S-transferases (GSTs), L-ascorbic acid oxidases (ASOs), peroxidases (PODs) and

oxidative

stress-related

pathways,

phytohormone

14


signaling

and

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catalases (CATs) were commonly induced in both two groups, but GTD (glutaredoxin)

308

genes in local group, and SOD (superoxide dismutase) and APX (ascorbate peroxidase)

309

genes in systemic group were triggered, respectively. Notably, several CAT and ASO

310

genes were obviously enhanced in local group, whereas several GST, POD and SOD

311

genes were intensely activated to scavenge ROS in systemic group (Supplementary

312

Figure S4b and Supplementary Table S5).

313

Comparative transcriptome analysis further revealed complex transcriptional

314

profiling of phytohormone signaling networks triggered by leaf-herbivory in tea

315

plants, including a prominent activation of JA, ET, ABA and MeSA signaling in local

316

group and a positive induction of JA, ABA and IAA signaling in systemic group

317

(Figure 4 and Supplementary Table S5). JA pathway was overall activated in local

318

group comprising up-regulated genes encoding lipoxygenases (LOXs), allene oxide

319

synthases (AOSs), allene oxide cyclases (AOCs), 12-oxo-phytodienoic acid

320

reductases (OPRs), jasmonate O- methyltransferase (JMT), JA ZIM-domain (JAZ)

321

and MYC proteins. Several LOX, AOS and MYC genes were also enhanced in

322

systemic group. Similarly, genes for ET signaling were significantly activated in local

323

group, including genes encoding S-adenosyl-L-methionine synthases (ADSs),

324

1-aminocyclopropane-1-carboxylate

325

1-aminocyclopropane-1-carboxylate oxidases (ACOs) and ethylene responsive factors

326

(ERFs). By comparison, ACS, ACO and ERF genes were up-regulated with less

327

amplification in systemic group. No gene responsible for SA biosynthesis and

328

perception was activated in both two groups, but 1 SAMT gene encoding salicylic acid

329

carboxyl methyltransferase for the conversion of SA to MeSA was up-regulated in

330

local group. For ABA pathway, 7 obviously elevated genes of zexanthin epoxidases

331

(ZEPs) and 9-cis-epoxycarotenoid dioxygenases (NCEDs) were identified in local

synthases

15


(ACSs)


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332

group. Although only 1 ZEP gene and 1 NCED gene were activated in systemic group,

333

the ZEP gene was up-regulated ~4700 folds (Log2Ratio values of 12.3). IAA pathway

334

was more remarkably activated in systemic group than in local group, such as

335

significantly

336

flavin-containing monooxygenase), 2 TIR (encoding transport inhibitor response) and

337

1 ARF (auxin response factor) genes with Log2Ratio values of 4.5~12.7.

elevated

expression

levels

displayed

in

1

YUCCA

(YUC

338

In addition, we identified a large amount of DEGs encoding 15 and 11 types of

339

transcription factors (TFs) related to insect resistance in local and systemic groups,

340

respectively. Several TF genes in MYC, WRKY, AP2/ERF, NAC, MYB and bHLH

341

families were dramatically enhanced in local group comparing to slight up-regulation

342

in expression levels of these genes in systemic group (Supplementary Figure S4c and

343

Supplementary Table S5). We further discovered that different transcripts for most of

344

above mentioned genes were induced between local and systemic signaling networks.

345

In sum, leaf-herbivory elicited different local and systemic signaling networks as

346

mediators for launching local defense response and enhancing systemic resource

347

reallocation.

348

Dynamic expression patterns of candidate genes in local and systemic

349

responses using qRT-PCR

350

To further analyze the dynamic spatiotemporal transcription patterns of the induced

351

local and systemic responses, several DEGs, which contained LOX, ACO, SAMT and

352

MYC genes related to phytohormone signaling, GST, ASO and POD genes for

353

scavenging of ROS, and chitinase and TPS genes for resistance, were selected to

354

detect the dynamic expression levels in treatment and control leaf/root samples from 3

355

to 24 h after leaf-herbivory using qRT-PCR. Almost all the candidate genes were 16


Page 17 of 43


356

up-regulated in local and systemic group after leaf-herbivory, but relatively stronger

357

activations were observed in local group (Figure 5), which was consistent with the

358

changing tendency from comparative transcriptome analysis. Notably, LOX3 gene

359

exhibited the strongest local and systemic responses, peaking with ~80-fold local

360

up-regulation at 6 h after leaf-herbivory and ~20-fold systemic up-regulation at 9 h

361

after leaf-herbivory, respectively. The relative expression levels of LOX1, LOX3,

362

MYC2, SAMT, GST1, ASO1, POD1, Chitinase1 and TPS5 genes peaked in local group

363

at 3~6 h after leaf-herbivory, but most of them peaked in systemic group at 6~9 h

364

after leaf-herbivory. However, the relative expression levels of ACO1 gene

365

maximized at 12~24 h in local group but at 3~6 h in systemic group after

366

leaf-herbivory (Figure 5). Therefore, the dynamic expression profilings of leaf and

367

root in response to leaf-herbivory were not synchronous.

368

Furthermore, the dynamic expression changes of these genes were also

369

investigated in treatment and control leaf/root samples from 3 to 24 h after

370

leaf-mechanical damage and compared with those induced by leaf-herbivory.

371

Mechanical damage elicited both local and systemic responses at transcription level.

372

Almost all of the relative expression levels of LOX3, SAMT, GST1, Chitinase1 and

373

TPS5 genes at 3~24 h after mechanical damage were much higher in local group,

374

whereas ACO1, MYC2 and ASO1 genes were more remarkably activated in almost all

375

of the time course in systemic group. Among them, LOX3 gene exhibited the

376

strongest response and peaked with ~25-fold up-regulation at 9 h after mechanical

377

damage in local group, but ACO1 gene was the most heavily affected in systemic

378

group and peaked with ~50-fold up-regulation at 12 h after mechanical damage. In

379

addition, the relative expression levels of most candidate genes, such as LOX1, MYC2,

380

ACO1, GST1, ASO1, POD1, Chitinase1 and TPS5 genes, peaked at 3~6 h after 17



381

mechanical damage in local group. However, the relative expression levels of LOX1,

382

LOX3, SAMT, POD1 and TPS5 genes peaked at 6~9 h after mechanical damage, and

383

MYC2, ACO1, GST1 and Chitinase1 genes reached the maximal expression at 12 h

384

after mechanical damage in systemic group (Figure 5). Therefore, the different

385

transcriptional reconfiguration of these candidate genes were also induced between

386

local and systemic groups by mechanical damage, and the local response also peaked

387

ahead of the systemic response after mechanical damage at transcriptional level.

388

However, the activated genes and the expression intensity were not identical between

389

the dynamic expression patterns of all candidate genes induced by leaf-herbivory with

390

those triggered by leaf-mechanical damage.

391

Root-mediated carbon dynamics induced by leaf-herbivory

392

To investigate the dynamic changes of carbohydrate metabolism in local and systemic

393

groups induced by leaf-herbivory on tea plants, several main carbohydrates and

394

organic acids, containing sucrose, D-fructose, D-glucose, citric acid and L-malic acid,

395

were evaluated using GC-MS method. Sucrose, one kind of disaccharide, is

396

decomposed into glucose and fructose in starch and sucrose metabolism pathway.

397

Leaf-herbivory elicited the significant declines of sucrose contents not only in local

398

group but also in systemic group. D-fructose is usually converted into intermediates

399

and involved into glycolysis pathway and fructose metabolism. D-fructose contents

400

were also significantly decreased in both local and systemic groups. D-glucose is one

401

kind of core hexose for glycolysis pathway and citrate cycle. D-glucose contents were

402

obviously consumed at 3~24 h in local group and at 12~24 h in systemic group after

403

leaf-herbivory. Citric acid and L-malic acid are the two organic acids in citrate cycle.

404

Their contents were markedly descended in systemic group whereas the contents of 18


Page 18 of 43

Page 19 of 43


405

citric acid were not significantly changed in local group (Figure 6). Furthermore, the

406

depletion of all the 5 primary metabolites in systemic group exhibited a trend of

407

gradual increase. As mentioned above, the remarkably transcriptional activation of the

408

catabolism of carbohydrate, containing starch and sucrose metabolism, glycolysis,

409

citrate cycle and fructose metabolism, were discovered in systemic group. However,

410

starch and sucrose metabolism, glycolysis and fructose metabolism were obviously

411

activated in local group except for citrate cycle (Figure 3). The results demonstrated

412

that the metabolic changes of the investigated carbohydrates and organic acids were

413

consistent with the transcriptional impact induced by leaf-herbivory.

414

Differential impact of leaf-herbivory on local and systemic

415

phytohormone levels

416

To further investigate the responsive phytohormones induced by leaf-herbivory, the

417

contents of JA, JA-Ile, SA and ABA were determined in CKL, EL, CKR and ER

418

samples from 3 h to 24 h after leaf-herbivory using HPLC-triple Quadrupole Mass

419

analysis (Figure 7). Leaf-herbivory exerted the strongest effect on JA levels, which

420

were prominently elevated in almost the entire time course in both EL and ER

421

samples than the controls. Comparing to the respective controls, JA levels in EL

422

sample rapidly increased and peaked (~890 ng·g-1 fresh weight, ng·g-1 FW) at 9 h after

423

leaf-herbivory, but JA levels in ER sample promptly enhanced and peaked (~600

424

ng·g-1 FW) at 3 h after leaf-herbivory. Similarly, JA-Ile levels were induced to elevate

425

in damaged leaves in all of the time course, and they were also fastly increased to the

426

maximum at 3 h after leaf-herbivory in undamaged roots. Completely different with

427

the dynamic accumulation of JA and JA-Ile, SA levels were induced to overall decline

428

in the time course in both local and systemic groups, and the bigger decreasing 19



429

amplitude in systemic group was found. ABA levels were induced to change in a

430

pattern of ′decreasing-increasing-decreasing′ and reach the highest level at 9 h after

431

leaf-herbivory in local group, whereas they were triggered to vary in a gradual and

432

continuous increasing pattern and peak at 6 h after leaf-herbivory in systemic group.

433

The results exhibited that all the investigated phytohormones were disturbed in

434

specific patterns by leaf-herbivory, and the levels of the same phytohormone were

435

also triggered to alter in the different modes between local and systemic groups. The

436

changes of JA, JA-Ile, SA and ABA at metabolic level were consistent with the

437

expression changes of genes related to their signaling pathways at transcriptional

438

level.

439

DISCUSSION

440

In this study, using transcriptomic and phytochemical analysis, we compared the local

441

and systemic responses and the related phytohormone signaling triggered by tea

442

geometrids attack on leaves of tea plants, and investigated the potential role of roots

443

in response to leaf-herbivory. Comparative transcriptome analysis demonstrated the

444

occurrence of tissue-specific transcriptome reconfiguration involved in perception of

445

stimuli, signal transduction, metabolism and defense between aboveground and

446

belowground after leaf-herbivory in tea plants. Dynamic expression pattern analysis

447

supported this discovery, and further suggested the time course of leaf and root

448

responses were not synchronous. Our results were consistent with the previous studies

449

in maize, cabbage and tobacco.15-18 Therefore, in response to leaf-herbivory, not only

450

herbaceous plants but also woody plants reprogramme their transcriptomes in order to

451

reconfigure their physiologies in both aboveground and belowground tissues for

452

adaptation of environmental stimuli. In addition, inducements of insect herbivory 20


Page 20 of 43

Page 21 of 43


453

usually contain chemical elicitor from the oral secretion and mechanical damage from

454

chewing of herbivores. By comparison of the dynamic expression patterns of

455

candidate genes, we observed that both local and systemic responses were induced by

456

leaf-herbivory and mechanical damage, and the induced systemic responses basically

457

appeared later than the induced local responses after the two treatments. However, the

458

activated genes and the expression intensity in both local and systemic groups were

459

not identical between leaf-herbivory and mechanical damage treatments. Our analysis

460

confirmed that the dynamic expression patterns of the candidate genes induced by

461

leaf-herbivory and mechanical damage was different. It suggested the effect from

462

leaf-herbivory by geometrids was not only caused by mechanical damage, chemical

463

elicitor can be another important inducement for the generation of local and systemic

464

responses of tea plants. The effects on tea plants of chemical elicitor and mechanical

465

damage should be further investigated and compared in the future to deeply

466

comprehend their different mechanisms.

467

Local defense response in damaged tea leaves induced by geometrid attack has

468

been detailed studied, and the enhancement of the biosynthesis of secondary

469

metabolites has been testified at transcriptional and metabolic levels. Our and Wang’s

470

previous work elucidated that secondary metabolism play important roles for local

471

defense response induced by geometrid attack at transcriptome level.28, 29 Cai et al.

472

revealed that geometrid attack induced the generation of abundant volatiles containing

473

a large amount of terpenoids and GLVs from the attacked tea leaves.30 Recently,

474

Wang et al. provided further metabolomic evidence that a plethora of secondary

475

metabolites, such as flavonoids, involved in defense against tea geometrids could be

476

significantly induced in damaged tea leaves.31 However, we have hardly known the

477

mechanism of resource investment for secondary metabolism in tea plants under 21



478

heavy herbivory before this work. Here, a deep comprehension of the inducible

479

defense against tea geometrid attack was acquired from the perspective of systems

480

biology for the first time. By deciphering the responsive transcriptome profiling of

481

primary and secondary metabolism, the activation of a large scale of genes for the

482

biosynthesis of secondary metabolites was also observed, which was consistent with

483

the above mentioned previous work. However, secondary metabolism was uniquely

484

activated in local response but not in systemic response, whereas a mass of activated

485

primary metabolism genes were enriched in the catabolism of local and systemic

486

carbohydrates, especially in more significant depletion of systemic carbon reserves.

487

Carbohydrate dynamics analysis further provided evidence that leaf-herbivory can

488

significantly reduce nonstructural carbohydrates not only in damaged leaves but also

489

in undamaged roots, supporting that the potential role of root was as a resource

490

mediator to improve the deployment of carbon reserves for more effective leaf

491

defense. It indicated that tea plants employed a root-mediated resource-based

492

resistance strategy in response to leaf-herbivory. Similarly, some studies reported that

493

more resources were imported into attacked leaves to support plant defenses.10, 11, 39-41

494

On the contrary, some attacked plants may employ herbivory-induced resource

495

sequestration strategy to increase carbon transport from damaged leaves to the roots

496

for reinforce of plant tolerance and regrowth capacity.8, 41-43 Recent studies reported

497

that some herbivore-attacked plants have evolved mixed tolerance-defense strategies

498

to spatially and temporally resist diverse herbivore attackers.44-47 Therefore, different

499

plant species may adopt different strategies for tolerance-defense trade-offs against

500

herbivore stress. In the future, the carbon reallocation and reutilization should be

501

further investigated for deep understanding of the mechanism of tolerance-resistance

502

trade-offs in tea plants. 22


Page 22 of 43

Page 23 of 43


503

In the present study, both local and systemic responses were activated by E.

504

oblique attack on leaves of tea plants via intricate tissue-specific signaling networks,

505

which was supported by evidence from comparative transcriptome analysis,

506

phytohormone

507

phytohormone-related genes. We identified a large amount of activated genes

508

involved in perception of herbivore elicitors, Ca2+ signaling, MAPK signaling, ROS

509

elimination, phytohormone signaling and activation of TFs, but there was hardly little

510

overlap of gene transcripts between local and systemic groups. JA was identified as a

511

crucial phytohormone for mediating local and systemic responses against

512

leaf-herbivory due to a remarkable elevation on JA levels and the expression levels of

513

the key LOX genes involved in JA biosynthesis in local and systemic groups. This

514

result was consistent with many studies of plant-herbivore interactions.4, 9, 10, 14, 16, 21, 48

515

In addition, we found that LOX genes peaked at 3~6 h after herbivory, and JA level

516

peaked at 9 h after herbivory in attacked leaves. Notably, LOX genes peaked 6~9 h

517

after herbivory, but JA level peaked at 3 h after herbivory in unattacked roots. It

518

indicates that E. oblique attack triggers not only local JA accumulation from de novo

519

biosynthesis, but also a putatively mixed pattern of systemic JA accumulation

520

containing de novo biosynthesis of JA and JA transport from leaves to roots,

521

suggesting JA as a long-distance signal to initiate root systemic response.

quantification

and

dynamic

expression

analysis

of

522

In spite of JA signaling, several other phytohormones were discovered to

523

participate in the induced local and systemic signaling networks. Transcriptional

524

activation of ET signaling pathway exhibited stronger in local leaves, which was

525

different from the induction of ET signaling in maize roots by leaf-herbivory.16 The

526

activation of ACO gene appeared latter than that of LOX and MYC genes and the

527

increasing of JA, suggesting that the induced JA signaling may promote de novo 23



528

biosynthesis of ET in damaged leaves. The suppression of SA were opposite to the

529

activation of JA or ET in both local and systemic signaling networks, which may be

530

explained by antagonistic interplay of JA/ET-SA that is generally conserved among

531

various monocot and eudicot species.49 ABA has been identified as a

532

herbivore-triggered systemic signal to induce root-to-leaf defense response in maize

533

plants.50 Here, although the local and systemic activation of ABA signaling genes

534

were elicited, but ABA levels were basically suppressed in local group and were

535

elevated in systemic group. We speculate ABA may be a putative leaf-to-root signal.

536

ABA biosynthesis in damaged leaves and the subsequent transport to roots may be

537

induced by leaf-herbivory, and de novo biosynthesis of ABA in roots also occurred. A

538

predominant activation of IAA biosynthesis and perception pathway in systemic

539

group suggested that IAA signaling were required for the induced systemic responses.

540

Machado et al. recently reported that auxin was rapidly induced by herbivory and

541

regulated a subset of systemic, jasmonate-dependent defenses in roots.10, 24 However,

542

the occurring time of systemic auxin signaling and the crosstalk interactions of IAA

543

with JA or ABA in tea plants need further research.

544

In this study, leaf-herbivory by E. oblique attack triggered differential

545

transcriptome reconfiguration between local and systemic responses in tea plants.

546

Comparative transcriptome analysis and carbohydrate dynamics analysis revealed that

547

a root-mediated resource-based resistance strategy was employed by tea plants in

548

response to leaf-herbivory. Phytohormone quantification and dynamic expression

549

analysis of phytohormone-related genes demonstrated the crucial role of JA and the

550

involvement of JA, ET and ABA in local signaling networks but JA, ABA and IAA in

551

systemic signaling networks (Table of Contents Graphic). This work provides new

552

insights into the intricate molecular mechanisms of leaf-to-root communication acting 24


Page 24 of 43

Page 25 of 43


553

as the mediators of induced local and systemic responses against leaf-herbivory, and

554

helps to extend our knowledge of the inducible defense strategies employed in tea

555

plants from the perspective of systems biology.

556

ABBREVIATIONS USED

557

ACO,

558


559

synthase; AOC, allene oxide cyclase; AOS, allene oxide synthase; ARF, auxin

560

response factor; ASO, L-ascorbic acid oxidase; APX, ascorbate peroxidase; BSTFA,

561

N, O-Bis-(trimethylsilyl)-trifluoroacetamide; LOX, lipoxygenase; Ca2+-ATPase,

562

calcium-transporting ATPase; CAT, catalase;

563

B-like protein; CBLI, CBL-interacting protein; CBP, calcium-binding protein; CDPK,

564

calcium-dependent protein kinase; COG, orthologous group; CS, cellulose synthase;

565

DEG, differentially expressed gene; ET, ethylene; ERF, ethylene responsive factor;

566

FDR, false discovery rate; FPKM, fragments per kilobase of exon model per million

567

mapped reads; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLV, green

568

leaf volatile; GO, Gene ontology; GR, glutathione reductase; GST, glutathione

569

S-transferase;

570

receptor-like kinase; MAPK, mitogen-activated protein kinase; MRP, multidrug

571

resistance protein; NCED, 9-cis-epoxycarotenoid dioxygenase; JA, jasmonic acid;

572

JAZ,

573

12-oxo-phytodienoic acid reductase; PI, protease inhibitor; PPO, polyphenol oxidase;

574

POD, peroxidase; PR, pathogenesis-related protein; PRR, pattern-recognition receptor;


JA

GTD,

glutaredoxin;

ZIM-domain;

JMT,

synthase;

oxidase;

ADS,

ACS,

S-adenosyl-L-methionine

CAM, calmodulin; CBL, calcineurin

LRR-RLK,

jasmonate

leucine-rich

O-

25


repeat-containing

methyltransferase;

OPR,


575

RK, receptor kinase; ROS, reactive oxygen species; SA, salicylic acid; SAMT,

576

salicylic acid carboxyl methyltransferase; SOD, superoxide dismutase; TF,

577

transcription factor; TMCS, trimethylchlorosilane; TIR, transport inhibitor response;

578

XTH, xyloglucan endotransglucosylase/hydrolase; YUCCA, YUC flavin-containing

579

monooxygenase; ZEP, zexanthin epoxidase; β-Gluc, β-1,3-glucanase.

580

FUNDING

581

This work received financial supports from the National Natural Science Foundation

582

of China (31300578), the National Key Research and Development Program of China

583

(2018YFD1000601), the project of the Youth Elite Supporting Plan in Universities of

584

Anhui Province (gxyq2018003) and the Doctoral Science Foundation of Anhui

585

Agricultural University (wd2016-02). These funders had no role in study design, data

586

collection and analysis, decision to publish, or preparation of the manuscript.

587

AUTHORS' CONTRIBUTIONS

588

CLW and HY conceived and designed the experimental plan. HY, YNW and LBL

589

participated in sample collection and experiments. JQW performed the analysis of

590

phytohormones. HY, YNW, FDL and YXH analyzed and interpreted the sequence

591

data. HY and YNW drafted the manuscript. HY, JQW and CLW revised the

592

manuscript.

593

CONFLICT OF INTEREST

594

The authors declare that they have no conflict of interest.

595

SUPPORTING INFORMATION 26


Page 26 of 43

Page 27 of 43


596

Availability of supporting data

597

The Illumina RNA-Seq data generated from damaged leaves and undamaged roots

598

from tea plants (C. sinensis) attacked by E. oblique and the control leaves and roots

599

are available in the NCBI SRA (http://trace.ncbi.nlm.nih.gov/Traces/sra) with

600

accessions SRR7758794–SRR7758804. Additional supporting information may be

601

found in the online version of this article.

602

Supplementary Figure S1. Characterization of homology search of the assembled C.

603

sinensis unigenes against the NR database. (a) The E-value distribution of the

604

alignment results of C. sinensis unigenes. (b) The similarity distribution of the

605

alignment results of C. sinensis unigenes. (c) The species distribution of the alignment

606

results of C. sinensis unigenes.

607

Supplementary Figure S2. Histogram presentation of clusters of orthologous group

608

(COG) classification of C. sinensis unigenes.

609

Supplementary Figure S3. Gene Ontology classification of C. sinensis transcriptome.

610

Gene Ontology (GO) terms are summarized in three main categories: biological

611

process, cellular component and molecular function. The left and right Y-axes in log

612

scale indicate the percentage and the number of genes within a certain GO category,

613

respectively.

614

Supplementary Figure S4. DEGs related to protein kinase, Ca2+ signaling, ROS

615

pathway and related TFs in the local and systemic signaling networks elicited by

616

leaf-herbivore attack on tea plants. (a) DEGs related to pattern-recognition receptors, and

617

involved in Ca2+ signaling and MAPK signaling pathways. (b) DEGs involved in

618

oxidative stress-related pathways. (c) DEGs related to transcription factors (TFs). The

27



619

numbers of the induced genes in leaves and roots by leaf-herbivory were indicated in

620

numerals above and below the X-axes, respectively.

621

Supplementary Table S1. Primers of the candidate unigenes designed for qRT-PCR.

622

Supplementary Table S2. Summary of RNA-Seq outputs for the twelve libraries.

623

Supplementary Table S3. Length distributions of assembled contigs and unigenes from

624

each library of C. sinensis.

625

Supplementary Table S4. The significantly enriched KEGG pathway annotated in the C.

626

sinensis reference transcriptome.

627

Supplementary Table S5. Differentially expressed genes involved in signaling, ROS,

628

TFs and defense response pathways in leaves and roots in response to E. oblique attack

629

on leaves of tea plants.

28


Page 28 of 43

Page 29 of 43


630

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786

FIGURE CAPTIONS

787

Figure 1. Comparative analysis of local and systemic transcriptomes induced by

788

E. oblique attack on leaves of tea plants.

789

(a) The Venn diagram of the induced DEGs in local group (EL-vs-CKL) and systemic

790

group (ER-vs-CKR). Red and blue arrows indicate up-regulated and down-regulated

791

DEGs, respectively. (b) Comparison of the enrichment of KEGG pathways of DEGs

792

in local and systemic groups.

793

Figure 2. Differential transcriptional profiling of local and systemic defense

794

responses triggered by leaf-herbivore attack on tea plants.

795

(a) Phenylpropanoid pathway. (b) Flavonoid biosynthesis. (c) Biosynthesis of H

796

lignins. (d) Caffeine biosynthesis. (e) Terpenoid biosynthesis. (f) GLV biosynthesis.

797

(g) Cell wall remodeling and generation of resistant proteins. Expression data are

798

plotted as Log2 values.

799

Figure 3. The transcriptional impact on carbohydrate metabolism in local and

800

systemic groups triggered by leaf-herbivore attack on tea plants.

801

(a) The transcriptional impact on the subcategories involved in local carbohydrate

802

metabolism. (b) The transcriptional impact on the subcategories involved in systemic

803

carbohydrate metabolism. The colorized dots represent the subcategories involved in

804

carbohydrate metabolism. The X-axes indicate the number of up-regulated DEGs

805

related to the certain subcategory of carbohydrate metabolism. The Y-axes indicate

806

the significance of the DEGs related to the certain subcategory of carbohydrate

807

metabolism, which are plotted as -Lg (P-value) values.

808

Figure 4. Differential transcriptional profiling of the local and systemic

809

phytohormone signaling networks elicited by leaf-herbivore attack on tea plants. 33



810

The investigated phytohormone signaling networks contained JA, ET, SA, ABA and

811

auxin signaling pathways. Expression data are plotted as Log2 values.

812

Figure 5. The dynamic expression patterns of candidate genes involved in

813

phytohormone signaling, oxidative stress-related pathway and defense response.

814

The dynamic expression levels of the candidate genes in damaged leaves and undamaged

815

roots were analyzed at 3~24 h after leaf-herbivory or mechanical damage using qRT-PCR.

816

(a, b) LOX1, lipoxygenase 1. (c, d) LOX3, lipoxygenase 3. (e, f) MYC2, MYC

817

transcription factor 2. (g, h) SAMT, salicylic acid carboxyl methyltransferase. (i, j) ACO1,

818

1-aminocyclopropane-1-carboxylate oxidase 1. (k, l) GST1, glutathione s-transferase 1.

819

(m, n) ASO1, L-ascorbate oxidase 1. (o, p) POD1, peroxidase 1. (q, r) Chitinase1. (s, t)

820

TPS5, terpene synthase 5. All the candidate genes were analyzed in leaf and root samples

821

treated by leaf-herbivory and mechanical damage and the control samples. GAPDH was

822

used as internal control. The expression of the genes in control samples was set to 1.0.

823

Different letters mean significant difference (P < 0.05) between local and systemic

824

groups at the same time point after leaf-herbivory.

825

Figure 6. Quantitative analysis of the contents of main carbohydrates and

826

organic acids induced by E. oblique attack on leaves of tea plants.

827

The detected carbohydrates and organic acids contained sucrose, fructose, glucose,

828

citric acid and L-malic acid. Different letters mean significant difference (P < 0.05)

829

between treatment and control samples in the local group or the systemic group.

830

Figure 7. Quantitative analysis of the variations of phytohormone contents

831

induced by E. oblique attack on leaves of tea plants.

832

The detected phytohormones contained ABA, JA, JA-Ile, and SA. Different letters

833

mean significant difference (P < 0.05) between treatment and control samples.

34


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834

Table

835

root-mediated resource-based resistance mechanism employed by tea plant in

836

response to leaf-herbivory by E. oblique.

of

Contents

Graphic.

A

schematic

diagram

35


summarizing

the


Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Table of Contents Graphic

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Transcriptomic and Phytochemical Analyses Reveal Root-Mediated

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