Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(2): 195-207 (2018)

In-silico

identi cation of phytohormone pathway genes

Camellia sinensis

and expression analysis under

combined water and herbivore stress

Madhurjya Gogoi

1,2

*, Hemanta Saikia

, Sangeeta Borchetia

, Raj Narain Singh Yadav

and

Tanoy Bandyopadhyay

Department of Biotechnology, Tea Research Association, Tocklai Tea Research Institute, Jorhat-785008,

Assam, India

Centre for Biotechnology and Bioinformatics, School of Science and Engineering, Dibrugarh University,

Dibrugarh-786004, Assam, India

Department of Life Sciences, School of Science and Engineering, Dibrugarh University, Dibrugarh-786004,

Assam, India

ABSTRACT

Tea (Camellia sinensis) is a popular beverage worldwide. Abiotic and biotic stresses due to recent climate change have

signi cant effect on yield of tea. Plant hormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA)

and ethylene (ET) plays an important role in regulating plant defense responses to different kind of stresses. In this

study homologous phytohormone genes of ABA, JA, SA and ET pathway in tea plant were identi ed from the public

domain transcriptomic database and the expression of the rate-limiting genes of phytohormone pathway were ana-

lyzed in tea plants subjected to combined water and herbivore stress to understand the interaction among the stress-

induced phytohormone pathways genes. Vegetatively propagated TV1 clones of tea plant were subjected to three

level of water stress treatments: 1) well watered control 2) mild water stress 3) severe water stress for three months

and then infested with Hyposidra talaca (looper caterpillar). The constitutive expression (without infestation) of the

rate-limiting genes of ABA and ET pathway were positively regulated by water stress whereas JA and SA pathway

genes were negatively regulated. On looper caterpillar infestation (induced expression) water stressed plants showed

signi cant decrease in expression of the rate-limiting phytohormone genes except ACC synthase. Our study showed

that on herbivore infestation well watered plants have higher capacity to induce the phytohormone genes and water

195

ARTICLE INFORMATION:

*Corresponding Author: madhurjyag@gmail.com

Received 28

March, 2018

Accepted after revision 16

June, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.1/2

196

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Madhurjya Gogoi et al.

stress played a major role in regulation of gene expression than herbivore infestation. The presence of an initial water

stress not only affected the tea plant constitutive defense but also signi cantly altered the phytohormone defense

gene expression towards subsequent herbivore stress. The water stressed tea plant with weak induced expression of

defense associated phytohormone genes may be at a higher risk for incidence of pest and pathogen attack compared

to well watered plants.

KEY WORDS:

CAMELLIA SINENSIS

, EXPRESSION ANALYSIS, HERBIVORE INFESTATION,

HYPOSIDRA TALACA

IN SILICO

IDENTIFICATION;

PHYTOHORMONE PATHWAY

INTRODUCTION

Tea, Camellia sinensis is a major economic crop world-

wide and its young leaves are used for preparing bever-

age. Huge losses in tea leaf yield is incurred due to the

present climate change scenario. As the climatic change

event is expected to increase the incidence of water

shortage and outburst of insect population, the mono-

culture cultivation of tea may face severe crop loss in

recent future. Drought is one of the major abiotic stress

that in uences the quality and productivity of crops by

growth inhibition, increase in organic solutes concen-

tration and changes in the endogenous phytohormones

content (Wijeratne et al., 2007; Bhagat et al., 2010,

Aimar et al. 2011, Chen and Chen, 2012 and Anderegg

et al. 2015).

Biotic stressors such as insects and pathogens, also

contribute signi cantly towards the enormous dam-

age to the crops (Hammond-Kosack and Jones, 2000).

Plants have both inherent and adopted mechanisms to

cope with the environmental stresses by producing cer-

tain proteins and secondary metabolites that are toxic

or have repellant effect on the biotic agents (Rani and

Jyothsna, 2010; War et al., 2011a; War et al., 2011b; War

et al., 2012). Abiotic and biotic stresses in plants trigger

the activation of a number of phytohormone pathway

genes which simultaneously activate other intercon-

nected defense network to help plants sustain the stress

period (Fraire-Velá zquez et al., 2011). The primary phy-

tohormones abscisic acid (ABA), jasmonic acid (JA), sali-

cylic acid (SA), ethylene (ET) are involved as messengers

triggering the speci c defense pathways against envi-

ronmental stress and may act individually or in combi-

nations depending upon the stress perceived, (Atkinson

and Urwin, 2012, Verma et al., 2016; Wani et al., 2016).

ABA is produced in response to water-de cit stress

(Osakabe et al., 2013). Enhanced accumulation of ABA

in Arabidopsis thaliana seedlings has been reported

under drought conditions (Huang et al., 2008). Exog-

enous application of ABA delay wilting and is reported

to induce drought tolerance in plants (Lu et al., 2009).

ABA functions both synergistically and antagonistically

with JA, SA and ET signaling pathways which play a

dominant role during biotic stress (Chen and Yu, 2014).

Under the combination of abiotic and biotic stresses,

ABA mostly acts as an antagonist to JA/SA/ET mak-

ing the plant susceptible to disease and pathogen attack

(Rejeb et al., 2014). However, a positive interaction has

also been observed, whereby an increase in ABA level

under abiotic stress results in stomatal closure which

prevents the entry of biotic agents and protects the

plants from both biotic and abiotic stresses (Melotto et

al., 2006). SA, an endogenous growth regulator, induces

systemic acquired resistance (SAR) in plants against dif-

ferent pathogens, particularly microbes and serves as a

signal molecule by producing pathogenesis related (PR)

proteins (Gao et al., 2015; Verma et al., 2016).

SA is also involved in plant response to different abi-

otic stresses such as drought, temperature variations,

heavy metals and osmotic stress (Rivas-San Vicente and

Plasencia, 2011). JA, a key regulator of plant response

to pathogens and insects, is involved in both direct and

indirect defenses of plants to herbivory (Creelman and

Mullet, 1995). JA also participates in plant’s response to

drought and salinity (Riemann et al., 2015). ET, the gas-

eous phytohormone for defense, helps in both direct and

indirect response of plants to abiotic and biotic stresses.

The effects of ET can be transitory or long lived as its

biosynthesis shows a diurnal rhythm and controls its

own biosynthesis (Eyidogan et al., 2012; Gamalero and

Glick, 2012; Verma et al., 2016).

Depending on the type of stress perceived by the

plant, different signaling pathways are activated which

synergistically or antagonistically in uence the type of

response generated. The interactions among the different

signal transduction pathways are considered as crosstalk

between the pathways which helps the plants to sustain

the stress period (Rejeb et al., 2014). In the event of cli-

matic change where both the abiotic and biotic stress will

co-occur, it is largely unknown how the phytohormone

based plant defense network would behave as majority

of the studies so far considered single stress factor either

abiotic or biotic at a time. It also remain unpredictable

how the presence of an initial stress affects the plant

defense network on perception of a subsequent stress.

As the frequency and extent of drought as well as insect

infestation are projected to increase due climate change

it is essential to understand the response of plants to

combined stress conditions. Knowledge of the molecular

mechanisms underlying these effects is very limited. The

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IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES 197

Madhurjya Gogoi et al.

molecular study of the stress-related hormonal genes

and their interactions would help to understand the syn-

chronization of plant constitutive and induced defense

responses to insect infestation in plants under abiotic

stress. Tea plant faces water stress event round the year

and also plethora of insect infestation. Looper cater-

pillar infestation stand out to be the most destructive

insect infestation in terms of crop loss. The water stress

may change the overall metabolism of the tea plant and

alter its defense interaction with the biotic agents. It will

be vital to know the interaction of the phytohormone

defense gene network in perception of combined water

and herbivore stress.

In this study, a comparative genomics approach was

undertaken to mine the phytohormone pathway genes

of C.sinensis and the expression pattern of the rate lim-

iting genes of the four phytohormone pathway (ABA,

JA, SA, ET) was analyzed in a clone (TV1) of tea plant

which was subjected to different regime of water stress

(abiotic stress) treatment with subsequent insect infesta-

tion (looper caterpillar). This study will help to better

understand the stress-induced phytohormones defense

interaction at transcriptional level in tea plant under

combined water and herbivore stress.

MATERIALS AND METHODS

IN SILICO

IDENTIFICATION OF PHYTOHORMONE

PATHWAY GENES IN

C. SINENSIS

The genes or transcripts involved with ABA, JA, SA and

ET phytohormone pathway in C. sinensis were mined

from different databases using Arabidopsis sequences as

reference. The Arabidopsis full-length coding sequences

(CDS) were collected from the TAIR database (https://

www.arabidopsis.org/) and subsequently the sequences

were subjected to BLASTN with C. sinensis Expressed

Sequence Tags (EST), Transcriptome Shotgun Assembly

(TSA) and Non-Redundant (NR) nucleotide databases of

NCBI. The sequences showing signi cant similarity with

an E-value ≤ 1e

-15

were selected and assembled using

CAP3 program to remove redundancy and get consensus

sequences. Each of those sequences were screened for

the presence of open reading frame (ORF) using NCBI

ORF Finder (Wheeler et al., 2003) and sequences with

the longest ORF having both start and a stop codon

were sorted out. The sequences were further subjected to

BLASTX with NCBI NR (Non-Redundant) protein data-

base to con rm their annotation. Based on the BLAST

annotation and alignment results, the sequences found

similar to Arabidopsis reference sequences and nearby

plant species sequences were retained and others were

 ltered out. The best representative sequences were sub-

jected to blast and functional classi cation following

the Gene Ontology (GO) scheme using BLAST2GO suite

(Conesa and Götz, 2008). The transcripts were classi ed

into the major GO categories, namely, cellular compo-

nent, molecular function and biological process. Further

the rate-limiting gene sequences of the phytohormone

pathways were used in expression study.

MOTIF AND DOMAIN IDENTIFICATION

MEME and MAST programs (Bailey and Elkan, 1994;

Bailey and Gribskov, 1998) were used for the identi ca-

tion of the motif cluster present in the phytohormone

pathway gene sequences. MEME program performs motif

discovery on DNA, RNA or protein datasets. Whereas,

MAST program searches sequences for matches to a set

of motifs and sorts the sequences by the best combined

match to all motifs. The sequences from C. sinensis and

Arabidopsis were analyzed together for the easy identi-

 cation of common motifs between them. The motif dis-

covery mode was set to normal. The maximum number

of motif was set to 20. Domain search was performed

using CD-search tool available at the conserved domain

database (CDD) of National Center (https://www.ncbi.

nlm.nih.gov/Structure/cdd/wrpsb.cgi).

SELECTION OF GENE FOR qRT PCR AND

PRIMER DESIGN

The rate limiting gene/enzyme of each phytohormone

pathway was searched from literature references and

the expression of the corresponding genes was analyzed

across different treatments. For the ABA pathway, the

reaction catalyzed by 9-cis-epoxycarotenoid dioxyge-

nase (NCED) i.e- the oxidative cleavage of neoxanthin

is considered as the rate-limiting step (Tan et al., 1997;

Qin and Zeevaart, 1999) and chosen for our study. In the

ET biosynthesis pathway, the conversion of S-AdoMet

to 1-aminoacyclopropane 1-carboxylate (ACC) by ACC

synthase is taken as the rate-limiting step (Wang et al.,

2002). Allene oxide synthase (AOS), the  rst enzyme

in the branch pathway leading to the formation of JA

act as rate-limiting step in JA biosynthesis (Harms et

al., 1995; Sivasankar et al., 2000). In the SA pathway,

isochorismate synthase (ICS) acts as the rate-limiting

enzyme (Serino et al., 1995; Gaille et al., 2003).Thus

NCED gene for ABA, ACC synthase gene for ET, AOS

gene for JA and ICS gene for SA pathway was chosen

for gene expression analysis. The primers for the cor-

responding genes were designed using primer3 (http://

frodo.wi.mit.edu/primer3/) software. The primers were

designed as such that the product length was within

100-200 base pair. List of the primers used in the study

is provided in Supplementary Table 1.

Madhurjya Gogoi et al.

198

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EXPERIMENTAL SET UP AND SAMPLE

COLLECTION

Two years old vegetatively propagated TV1 clones of

tea plant were collected from the nursery of Tocklai Tea

Research Institute, TRA, Jorhat. The plants from nursery

were replanted in black poly-sleeves (18 cm diameter

and 23cm height) with  eld soil (sandy loam, pH 4.8-

5.1, bulk density 1.3-1.4 Mg m

-3

, single super phophate

0.5 kg. m

−3

of soil) and allowed to acclimatize for 30

days in natural environmental condition with suf cient

irrigation. After the acclimatization period the plants

were transferred to a polyhouse and were allowed to

acclimatize within the polyhouse for 10 days before

starting the stress experiments. The plants were covered

with nets for protecting it from external pest infestation.

Thereafter the plants were subjected to three level of

water stress treatment: 1) well-watered control; 2) mild

water stress; 3) severe water stress. The plants in each

drought stress level received same amount of water and

watered simultaneously. The well-watered control plants

received water every 3rd day such that the soil remained

constantly moistened. Mild drought stressed plants were

watered once the soil water content drops to 7-8% and

received 40-50 % of water supplied to the well-watered

control plants. The severe water stressed plants were

watered when the soil water content reaches 4-5 % and

received around 15–20% the amount of water supplied

to well-watered control plants. At the end of the three

months the plants were rehydrated with small amount of

water overnight in all the treatment and then subjected

to Hyposidra talaca (looper caterpillar) infestation.

Rehydration of plants was done to ensure that the

gene expression of plants re ect the effect of pulsed

water stressed treatment which is often faced by tea

plant in natural environment rather than continuous

drought. The leaves were collected before insect infesta-

tion i.e - at 0 hours (0 TPI), and after insect infestation

i.e - 24, 48 hours (24 TPI, 48 TPI) in all the treatments.

The leaves collected at 0 TPI were taken as undamaged

control. As only the non-damaged control tissue was

measured at the initial time point (time = 0 hrs), the

main effect of time represents time post herbivory infes-

tation or time post infestation and is designated at TPI.

For each treatment and for each time point there were

three biological replicates. Once the plants were used to

collect sample they were discarded and not used further

in the experiment. For all the treatment the third leaf of

tea plant from top was collected and immediately stored

at -80

C to prevent any enzymatic activity.

RNA ISOLATION AND cDNA PREPARATION

Total RNA was extracted from 100 mg of each sample

according to the protocol of Zaman et al., 2016. RNA

integrity was determined using a 1% agarose gel and

concentration was quanti ed using an Eppendorf Bio-

photometer (Eppendorf, Hamburg, Germany). After veri-

fying the integrity of the RNA, equal concentration of

RNA from each sample was used for  rst strand cDNA

preparation using QuantiTect Rev. Transcription Kit(Cat

No./ID:205311, QIAGEN, Germany).

QUANTITATIVE REAL-TIME PCR ANALYSIS

Four important genes known to be involved in the rate-

limiting step of the phytohormone biosynthesis pathway

(ABA, JA, ET, SA) in tea were selected based on the com-

parative in-silico identi cation of homologous genes. The

expression of the selected four genes was studied in tea

plant subjected to three level of water stress treatments

with subsequent insect infestation. Quantitative Real-

Time PCR was performed in a Roche Light Cycler 480 real

time machine (Roche, Germany) using QuantiTect SYBR

Green PCR Kit (Cat No./ID:204145, QIAGEN, Germany).

The reverse transcribed  rst strand cDNA of each sample

was used as template in the assay and ampli ed by gene-

speci c primers. The PCR was performed in 10 μl reaction

volume and prepared according to the protocol mentioned

in the kit manual. In short, 3.5 μl of supplied PCR grade

water was mixed with 0.5 μl of forward and reverse primer

and 5 μl of ‘QuantiTect SYBR Green I Master Mix’ to get

a  nal volume of 9.5 μl. Finally, 0.5 μl of template was

added. The relative expression levels of all the genes were

calculated using the ddCt method. The raw Ct values were

normalized against Ribulose-1, 5-bisphosphate carboxy-

lase/oxygenase housekeeping gene. The log 2 fold change

values for all the samples was calculated relative to well-

watered control sample at 0 TPI.

STATISTICAL ANALYSIS

The transcript relative expression values were log 2 fold

transformed and analyzed with a 3  3 (T x TPI) mixed

model analysis of variance (ANOVA) followed by post

hoc pairwise comparisons with Bonferroni adjustment

for multiple testing. “T” stands for water stress treat-

ment and “TPI” stands for time post infestation. All

the expression data are expressed as mean ± standard

deviation (SD). Each expression value is the mean of

three biological replicates. Data were analyzed using

IBMSPSS Statistics, Version 20.0.

RESULTS AND DISCUSSION

IDENTIFICATION AND ANALYSIS OF

PHYTOHORMONE PATHWAY GENES

Gene mining of the four phytohormone pathways (ABA,

JA, SA and ET) resulted in a large number of C. sinen-

Madhurjya Gogoi et al.

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IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES 199

sis homologues for each gene (Table1). CAP3 cluster-

ing removed the redundancy of the sequences, still a

signi cant number of homologues were retained after

clustering in many instances. In the ABA pathway few

genes like ABA 8’-hydroxylase, 9-cis-epoxycarotenoid

dioxigenase (NCED) etc. were represented by more than

one homologue of C. sinensis having signi cant similar-

ity and they possessed most of the common motifs pre-

sent in corresponding Arabidopsis reference sequences

(Table 1, Fig.1 (A, B)). Homologue gene mining of JA

pathway resulted in the identi cation of nine genes and

each gene best representative homologue is listed in

Table1. For the SA pathway a total of four genes were

mined but only three gene homologues were retained.

The C. sinensis homologues obtained for salicylic acid

carboxyl methyltransferase gene had an E-value >1e

and hence it was not considered for further study.

Two full-length sequences of ET pathway genes namely

1-aminocyclopropane-1-carboxylate synthase (ACCS)

and 1-aminocyclopropane-1-carboxylate oxidase

(ACCO) were obtained based on keyword search in the

NCBI NR nucleotide database.

Further the result of Blast2GO program with the

details of sequence similarity, GO classi cation, enzyme

list, InterPro scan domain etc. for the four pathway

genes are provided in Supplementary Table 2. MEME/

MAST search, for the putative functional and common

motif occurrence showed that most of the motifs are

conserved among the C. sinensis homologues and its

Arabidopsis counterpart except in few cases, where it

was seen that few motifs were missing in C. sinensis

homologues. This may be due to the presence of par-

tial transcript sequences of C. sinensis. Few representa-

tive genes displaying the occurrence of common motifs

between Arabidopsis and C. sinensis homologues are

shown in Fig.1 (A, B), Supplementary Fig. 1 (A-D) &

Supplementary Fig. 2 (A-D). The result of the functional

domain identi cation using CD-search tool showed the

presence of conserved domains between Arabidopsis and

Camellia sinensis homologues (Supplementary Table 3).

The presence of conserved common motif and domain in

sequential pattern between the phytohormone pathway

genes of C. sinensis and Arabidopsis clearly con rms

their identity and support the results of our comparative

genomics approach.

Gene expression of the rate-limiting genes 9-cis-

epoxycarotenoid dioxygenase (NCED), Allene oxide syn-

thase (AOS), 1-aminocyclopropane-1-carboxylate syn-

thase (ACC synthase) and Isochorismate synthase (ICS)

of ABA, JA, ET and SA biosynthesis pathway respec-

tively was studied at different time point in the three

water stress treatment with subsequent insect infesta-

tion. The relative gene expression values discussed here

are expressed in terms of log 2 Fold Change and the well

watered treatment at 0 TPI is taken as control for calcu-

lation of relative gene expression fold change for other

time points and treatments.

Considering the expression of rate limiting genes at 0

TPI (without insect infestation) the expression of NCED

gene of ABA pathway in case of mild and severe water

stress treatment was higher than well watered plants.

Mild water stress plant showed a log 2 FC value of 1.31

while severe stress plant showed a value of 1.62 (Fig.2

(A)). In mild water stress plants the AOS gene of JA

pathway had almost similar expression value with con-

trol and the difference was not statistically signi cant.

Whereas in severe water stress plants there was signi -

cant down regulation of AOS expression with a log 2

FC value of -2.49 compared to control plants (P< 0.05,

Fig.2 (B)). ICS gene of SA biosynthesis pathway showed

higher transcript accumulation in mild water stress plant

with a value of 2.84 whereas in severe stressed plants it

was down-regulated with a value of -4.04 compared to

control ( P< 0.05, Fig.2 (C)). In case of ET pathway, with

the increase in water stress intensity the expression of

ACC synthase gene also increased proportionally with a

value of 1.88 and 2.59 in mild and severe water stress

treatment respectively (Fig.2 (D)). The transcript expres-

sion at 0 TPI represent the constitutive expression of tea

plant and it mainly re ect the effect of the water stress

treatment on the expression of the phytohormone path-

way genes. The expression at 24 TPI and 48 TPI mainly

represent the induced expression of the tea plant after

insect infestation under different levels of water stress

treatment.

At 24 TPI, the expression of NCED gene in well-

watered plants increased signi cantly (log 2 FC: 2.13)

compared to control at 0 TPI. However the expression at

48 TPI was somewhat less (log 2 FC: 1.42) compared to

24 TPI. In the mild stress plants there was up-regulation

at 24 TPI followed by down-regulation at 48 TPI with a

value of -0.04. The severe stress plant showed a decline

in expression pattern with the increase of time (1.62 at

0 TPI, 1.38 at 24 TPI and 0.04 at 48 TPI). AOS gene

of JA pathway at 24 TPI showed signi cant induction

in expression (log 2 FC 3.05, P<0.05) of well-watered

plants. As the time elapsed the gene expression declined

at 48 TPI (Fig.2 (B)). However the expression was still

signi cantly higher compared to 0 TPI. Mild and severe

stressed plant followed the same trend with increase of

transcript accumulation at 24 TPI and then a decline at

48 TPI. The expression of ICS gene in case of control and

severe stressed plants increased at 24 TPI and decreased

at 48 TPI whereas in mild stressed plant the expression

declined both at 24 TPI and 48 TPI (Fig.2 (C)). Expres-

sion of ACC Synthase of ET pathway increased at 24 TPI

and then declined at 48 TPI in all the water stress condi-

tions (Fig.2 (D)). Two way ANOVA showed that for the

Madhurjya Gogoi et al.

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IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 1. Results of in-silico mining of Camellia sinensis phytohormone genes..

Arabidopsis

reference

sequence

accession

numbers

Sequence Name

Associated

Pathway

Total

No. of

Homologs

(EST + NR

+ TSA)

before

CAP3

Total

sequences

after CAP3

assembly

(Contig +

singleton)

Camellia sinensis

homologous sequences

retained after

processing

CAP3

(Contig,

singleton)

Number

Accession No.s

of Contigs &

singletons

Blast Annotation

BlastX similarity

percentage

and best hit

organism

AT4G19230,

AT2G29090,

AT5G45340,

AT3G19270

ABA 8’-hydroxylase

(ABA8ox)

Abscisic acid

pathway

22 5 ABA 8’-hydroxylase

(ABA8ox) [Singleton1,

Singleton2]

Singleton1 HP733304.1 ABA 8’-hydroxylase 86% Citrus

sinensis

Singleton2 HP764411.1 PREDICTED: abscisic acid

8’-hydroxylase 4

83% Vitis

vinifera

AT1G52400 ABA glucosidase 12 6 ABA glucosidase

[Contig1]

Contig1 KA279844.1,

KA279587.1,

HP733896.1,

GH710784.1,

GH710770.1,

FE942881.1

beta-glucosidase-like protein 99% Camellia

sinensis

AT2G27150 abscisic aldehyde oxidase

(ABAO)

6 1 abscisic aldehyde

oxidase (ABAO)

[Contig1]

Contig1 KA288987.1,

HP767578.1,

KA286688.1,

KA282538.1,

HP727479.1,

HP770168.1

PREDICTED: aldehyde oxidase

4-like

87% Vitis

vinifera

AT1G16540 molybdenum cofactor

sulfurase

3 2 molybdenum cofactor

sulfurase [Contig1]

Contig1 HP742704.1,

JK475554.1

PREDICTED: molybdenum

cofactor sulfurase-like

70% Vitis

vinifera

AT4G18350,

AT3G14440,

AT1G30100,

AT3G24220,

AT1G78390

9-cis-epoxycarotenoid

dioxigenase (NCED)

18 4 NCED [Singleton1,

Contig2]

Singleton1 HP727751.1 9-cis-epoxycarotenoid

dioxygenase 1

88% Diospyros

kaki

Contig2 HP765237.1,

BJ999395.1

putative 9-cis epoxycarotenoid

dioxygenase

88% Daucus

carota subsp.

sativus

AT1G67080 neoxanthin synthase

(NSY)

2 1 NSY[Contig1] Contig1 KA283257.1,

HP702818.1

neoxanthin synthase 73% Citrus

sinensis

AT1G52340 xanthoxin dehydrogenase

(XD)

7 6 XD[Contig1] Contig1 HP701207.1,

JK341963.1

short chain alcohol

dehydrogenase

79% Citrus

sinensis

AT5G67030 zeaxanthin epoxidase

(ZEP)

4 1 ZEP[Contig1] Contig1 HP760589.1,

HP739212.1,

HP727722.1

zeaxanthin epoxidase 1 82% Vitis

vinifera

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IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES 201

AT3G45140 13-lipoxygenase/LOX2 Jasmonic acid

pathway

20 5 LOX2[Contig1] Contig1 KA280187.1,

DY523322.1,

HP756288.1,

FE942952.1

lipoxygenase 99% Camellia

sinensis

AT4G16760,

AT5G65110,

AT1G06290,

AT3G51840,

AT2G35690

acyl-CoA oxidase 38 13 ACX[Contig4, Contig5] Contig4 FE943071.1,

KA288172.1,

KA280456.1,

HP709220.1,

HP724420.1 ,

HP740917.1,

HS399721.1,

KA280456.1

Peroxisomal acyl-coenzyme A

oxidase 1-like

70% Nicotiana

tomentosiformis

Contig5 HP754805.1,

KA298085.1,

KA300729.1

acyl-coenzyme A oxidase 2 85%

Brassica oleracea

var. oleracea

AT3G25760,

AT3G25770,

AT3G25780

allene oxide cyclase 46 5 AOC[Singleton1,

Singleton2]

Singleton1 HP769131.1 allene oxide cyclase 74% Camellia

sinensis

Singleton2 KA282377.1 allene oxide cyclase 99% Camellia

sinensis

AT5G42650 allene oxide synthase

(CYP74A1)

4 3 AOS[Contig1] Contig1 KA283779.1,

HP717071.1

cytochrome P450 allene oxide

synthase

77% Populus

trichocarpa

AT5G07010 hydroxyjasmonic acid

sulfotransferase

5 4 hydroxyjasmonic

acid sulfotransferase

(AtST2a)[Contig1]

Contig1 KA297677.1,

HP755751.1

PREDICTED:  avonol

sulfotransferase-like

74% Vitis

vinifera

AT1G19640 jasmonic acid carboxyl

methyltransferase

1 NA NA KA286401.1 KA286401.1 Jasmonate O-methyltransferase,

putative

67% Ricinus

communis

AT2G46370 jasmonic acid-amino acid

synthase

6 4 JAR[Contig1] Contig1 HP705426.1,

KA286035.1

JAR1-like protein 80% Nicotiana

attenuata

Keyword

OPC-8:0-CoA ligase NA NA NA NA FS957551.1 OPC-8:0-CoA ligase NA

AT1G20510 OPC-8:0-CoA ligase 6 4 OPCL1[Contig1] Contig1 HP750023.1

,KA293135.1,

KA286488.1

4-coumarate--CoA ligase-like 5 76%

Sesamum

indicum

AT2G06050 OPDA reductase 9 2 OPR3[Contig1] Contig1 HP743074.1,

KA280376.1,

HS399901.1,

HP725651.1,

HP746582.1,

HS398253.1,

HS398153.1,

KA302045.1

PREDICTED:

12-oxophytodienoate reductase

82% Vitis

vinifera

Madhurjya Gogoi et al.

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IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

NCED gene expression there was signi cant main effect

for time post infestation and interaction effect between

water stress treatment and time post infestation whereas

for AOS, ICS and ACC synthase gene expression both

the main effect (water stress treatment, time post infes-

tation) and there interaction effect was signi cant (Fig

2 A- D). The homogeneity and speci city of the sin-

gle PCR product was determined by the melting curve

and melting peak of the four analyzed genes and are

provide in Supplementary Fig. 3 (A-D), Supplementary

Fig. 4 (A-D) For supplementary data please see:https://

drive.google.com/drive/folders/1qVqzAqW1IEg-

kKnh6RA75zkdVIUxx0xH?usp=sharing.

It can be seen that as the water stress (0 TPI) increased

the transcript abundance of NCED gene also increased

(Fig.2 (A)). Water stress is known to increase the expression

of NCED gene followed by accumulation of ABA (Shino-

zaki and Yamaguchi-Shinozaki, 2007; Wang et al., 2009).

The function of ABA in the control of stomata closure and

the responses to abiotic stress is well-established (Mittler

and Blumwald, 2015). ABA integrates various stress sig-

nals and is known to controls stress responses during water

de cit stress (Raghavendra et al., 2010; Ye et al., 2012).

After insect infestation the expression of NCED gene in

well watered plant showed higher induction than mild and

severe stressed plants (24 TPI) and then there was a drop in

transcript level at 48 TPI for all treatment with well watered

plant retaining the highest transcript abundance.

When the expression folds were analyzed for statisti-

cal signi cance main effect of water stress treatment (T)

on NCED gene expression was not statistically signi cant

(P> 0.05). However the main effect of time post infesta-

tion (TPI) and interaction effect (TxTPI) was signi cant

(P<0.05). Herbivore infestation and the interaction of water

stress treatment and time post infestation signi cantly

regulated the expression differences. The strong induc-

tion of NCED expression in well-watered and mild stressed

plants along with the increase in AOS gene expression of

JA pathway at 24 TPI (Fig. 2(A) , Fig.2(B)) strongly sug-

gest the synergistic role of ABA and JA signaling pathway

genes in plant herbivore defense. On the other hand severe

stressed plant produced a smaller induction for both NCED

and AOS gene. It is very likely that water stress severity

negatively affected the induced response of the ABA and

JA biosynthesis pathway genes on herbivore attack. ABA

have been reported to interact with JA signaling and ena-

bles N. attenuataplants to mount a full defense response

against chewing herbivores (Dinh and Baldwin, 2013).

The expression of AOS, JA pathway rate-limiting

gene, was signi cantly down-regulated in severe water

stressed plants with a value of -2.49 compared to

well watered control plants (0 TPI, P<0.05, Fig. 2 (B)).

Irrespective of water treatment on herbivore infesta-

tion AOS gene expression increased at 24 TPI for all

AT1G18870,

AT1G74710

isochorismate synthase Salicylic acid

pathway

5 2 ICS[Contig1] Contig1 HP734410.1,

KA297692.1,

isochorismate synthase,

putative

69% Ricinus

communis

AT2G23620,

AT2G23600,

AT2G23560,

AT4G37150

methyl salicylate esterase 13 6 Methyl_salicylate_

esterase [Singleton1]

Singleton1 KA281093.1 PREDICTED: polyneuridine-

aldehyde esterase

63% Vitis

vinifera

AT2G43840,

AT2G43820

salicylic acid

glucosyltransferase

14 8 salicylic acid

glucosyltransferase

[Singleton1, Contig2]

Singleton1 KA286158.1 PREDICTED: UDP-

glycosyltransferase 74F2

71% Vitis

vinifera

Contig2 HP768085.1,

KA296966.1

PREDICTED: UDP-

glycosyltransferase 74E1-like

80% Vitis

vinifera

Keyword

1-aminocyclopropane-1-

carboxylate synthase

Ethylene

pathway

NA NA NA NA EF205149.1 1-aminocyclopropane-1-

carboxylate synthase

Keyword

1-aminocyclopropane-1-

carboxylate oxidase

NA NA NA NA DQ904328.1 ACC oxidase NA

Madhurjya Gogoi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

IN-SILICO

IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES 203

FIGURE 1. (A) Schematic representation of occurrence and distribution of motifs in the ABA

8’-hydroxylase sequences of Arabidopsis and Camellia sinensis - MEME program output (B)

Schematic representation of motifs and sorting of Arabidopsis and Camellia sinensis ABA

8’-hydroxylase sequences by the best combined match to all motifs - MAST program output.

FIGURE 2. Expression of 9-cis-epoxycarotenoid dioxygenase (NCED) gene of ABA pathway (B)

Expression of Allene oxide synthase (AOS) gene of JA pathway (C) Expression of Isochoris-

mate synthase (ICS) gene of SA pathway (D) Expression of 1-aminocyclopropane-1-carboxylate

synthase (ACC synthase) gene of ET pathway. Signi cant main effect of water stress treatment

and time post infestation are indicated by T and TPI respectively, while signi cant interaction

are indicated by TxTPI (P<0.05). All the expression data are calculated relative to well watered

control sample at 0 TPI and are expressed as mean ± standard deviation (SD). Each expression

value is the mean of three biological replicates.

the plants with control showing maximum transcript

abundance. It is noteworthy to mention that severe

stressed plant had maximum induction value when the

induced expression fold change of individual treatment

at 24 TPI is calculated relative to its own expression

at 0 TPI. Wound induced elevated level of AOS gene

expression was found to correlate with the increase in

endogenous JA content (Wilmowicz et al., 2016). JA is

mainly involved in biotic stress response and its role

biotic defense is well established. The detailed molecular

mechanisms of the role of jasmonates for drought stress

signaling are still unclear (Riemann et al., 2015).Plant

Madhurjya Gogoi et al.

204

IN-SILICO

IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

responses to combined abiotic stresses and biotic stress

are largely controlled by different signaling pathways

that may interact and inhibit one another (Suzuki et

al., 2014). In our study the expression of AOS gene of

JA biosynthesis pathway is clearly suppressed in severe

water stressed plants (0 TPI, 24 TPI). High transcript

accumulation of AOS gene in controls and mild water

stressed plants compared to severe stressed plants indi-

cates that water availability play an important role in

stronger constitutive and induced JA pathway defense.

Plants under increasing drought stress may be at dan-

ger of herbivore infestation with lower activation of JA

pathway defense. In some plants, it has been reported

that a speci c abiotic stress enhanced the resistance of

plants to biotic stress (Rouhier and Jacquot, 2008). How-

ever, in most cases, prolonged exposure of plants to abi-

otic stresses, such as drought resulted in the weakening

of plant defenses (Mittler and Blumwald, 2010). Plants

under combinations of abiotic and biotic stresses may

prioritize responses to address the potentially more dam-

aging abiotic stress (Atkinson et al., 2013).

Increase in ICS gene expression at 0 TPI in mild water

stress points towards the role of SA pathway in moderate

water stress condition. SA signi cance has been increas-

ingly recognized in enhanced plant abiotic stress-toler-

ance via SA-mediated control of major plant-metabolic

processes (Khan et al., 2015). Studies extensively found

and reviewed the role of SA pathway in the improve-

ment of plant abiotic stresses tolerance such as drought

(Horváth et al., 2007;Pal et al., 2013; Fayez and Bazaid,

2014; Miura and Tada, 2014). In induced response (24 TPI)

well watered plant maintained higher ICS gene expres-

sion followed by mild and severe water stressed plant. It is

known that upon insect attack usually two signaling path-

ways Salicylic acid (SA) and Jasmonic acid (JA), mediate

plant responses. Severe water stress negatively affected

the ICS gene expression at constitutive and induced level.

The lower expression of SA biosynthesis pathway gene

in water stress tea plant may increase their susceptibil-

ity to herbivores and necrotrophic pathogens. A signi -

cant amount of literature mentioned that the induction of

the SA signaling pathway suppresses JA signaling (Niki

et al., 1998; Preston et al., 1999; Koornneef et al., 2008a,

2008b).

However, in our study, we have seen a completely dif-

ferent picture where SA and JA associated gene expres-

sion increased co-currently in control and mild stressed

plants. Whereas in case of severe stressed plant both the

genes were signi cantly down-regulated. Thus JA and SA

pathway associated gene expression may not always act

antagonistically and may act in synchrony according to

the type and severity of stress it is undergoing. In tea

cultivation, water stress severity is an important factor

which needs to be taken care to avoid disastrous pest and

pathogen attack due to weakening of SA and JA associ-

ated plant defense.

ACC synthase expression showed maximum peak in

sev ere stressed plants at 0 TPI. The increased constitu-

tive expression in water stressed plants may be asso-

ciated with ET signaling role in osmotic stress adjust-

ment. ET signaling is known to act as an important

controller of the hormone-regulated defense pathways

in biotic stress (Broekgaarden et al., 2015) as well as

helping plants to adjust to drought stress (abiotic stress)

by increasing the compatible solutes accumulation (Cui

et al., 2015). Pairwise comparison showed that induced

expression value was not signi cant between control

and severe stressed plant (P >0.05, Fig.2 (D)). However it

was signi cant between control and mild stressed plant

(P< 0.05, Fig.2 (D)). When the fold change at 24 TPI in

all treatment is calculated relative to its own expression

at 0 TPI the increase in well-watered fold induction in

maximum. Thus the increase of ACC synthase expression

in response to herbivore infestation points towards ET

signaling role in biotic stress signaling and well-watered

plant have higher capacity of transcript induction on

insect infestation. Ethylene transcripts mediated role in

plant stress and pathogen responses have been already

reported in literature (Abeles et al., 1992; O’Donnell et

al., 1996). In rice plant, ACC oxidase and ACC synthase

transcript up regulation in response to feeding by the

brown planthopper, Nilaparvata lugens (Stal) have been

documented (Zhang et al., 2004).

Overall from our study it has been seen that water

stress positively affected ABA and ET pathway genes

with increase in constitutive expression. However JA and

SA pathway genes known to be involved with herbivore

and pathogen defense signaling were negatively affected

in severe stressed plants. In case of induced expression

(24 TPI), severe water stressed plants showed signi cant

decrease in expression of the rate-limiting phytohor-

mone genes compared to well water plants except ACC

synthase gene. The crosstalk among the phytohormone

pathway transcriptional defense in combination with

other intrinsic defense mechanism will ultimately gov-

ern the of tea plant response.

The presence of abundant antagonistic or synergistic

interactions among the pathways provide the plant with an

extensive regulatory potential for the activation of speci c

defenses (Vos et al., 2013). When combination of water and

herbivory stress was applied, it seems to be most likely that

plant transcriptional defense machinery was largely con-

trolled by water stress severity and herbivory stress regula-

tion of phytohormone genes was overpowered by water

stress gene regulation. Thus our results clearly shows that

well-watered plants on insect infestation will have higher

capacity to induce the expression of phytohormone genes

than water stressed plants.

Madhurjya Gogoi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

IN-SILICO

IDENTIFICATION OF PHYTOHORMONE PATHWAY GENES 205

CONCLUSION

It can be concluded that the presence of an initial water

stress not only affected the tea plant constitutive defense

but also signi cantly altered the phytohormone defense

gene expression towards subsequent herbivore stress. On

prolonged drought events tea plant with weak induced

defense may face higher incidence of pest and pathogen

attack.

CONFLICT OF INTEREST

The authors declare that they have no con ict of

interest.

ACKNOWLEDGEMENTS

The authors are grateful to the Director, Tocklai Tea

Research Institute (TTRI), Tea Research Association

(TRA) for providing the necessary facilities to conduct

the research work. The authors also acknowledge the

support and facilities provided by Director In-Charge,

Centre for Biotechnology and Bioinformatics, School of

Science and Engineering, Dibrugarh University for car-

rying out the research activity.

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