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Molecular cloning and characterization of hydroperoxide lyase gene in the leaves of tea plant (Camellia sinensis) Wei-Wei Deng, Yi-Lin Wu, Ye-Yun Li, Zhen Tan, and Chao-Ling Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05748 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on February 17, 2016

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Journal of Agricultural and Food Chemistry

Molecular cloning and characterization of hydroperoxide lyase gene in the leaves of tea plant (Camellia sinensis)

Wei-Wei Deng, Yi-Lin Wu, Ye-Yun Li, Zhen Tan, Chao-Ling Wei * State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China

*

Corresponding author; Tel/fax: +86 551 65786765

Email address: [email protected] (C.-L. Wei)

Running Title: Hydroperoxide lyase (HPL) from Camellia sinensis

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ACS Paragon Plus Environment


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ABSTRACT: Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the

2

biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was

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known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was

4

isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to

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herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by

6

RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST

7

searches showed high homology to HPL sequences from other plants. The His-tag

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expression vector pET-32a(+)/CsHPL was constructed and transferred into

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Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E.

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coli was about 60 kD. The enzyme activity of CsHPL was 0.20 µmol·min-1·mg-1.

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Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea

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plants after Ectropis obliqua attack, suggesting that it may be an important candidate

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for defense against insects in tea plants.

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KEYWORDS: Camellia sinensis• hydroperoxide lyase • Ectropis obliqua •

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suppressive subtractive hybridization (SSH) • induced defense response

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INTRODUCTION

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Tea plant (Camellia sinensis), an evergreen shrub, is one of the most important

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economic plants in the word. Tea plants produce specific and healthy compounds

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such as caffeine, theanine and catechins. Many reports exist on their content, their

20

biosynthesis and the healthy function of these compounds. As an evergreen woody

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plant, the cultivation of tea plant needs long time and is very susceptible to biotic

22

(e.g., insect attack) and abiotic (cold and drought, etc) stresses.

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Plant defense mechanisms include both direct and indirect defenses. The

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lipoxygenase (LOX) pathway, an important indirect defense pathway, includes two

25

biosynthetic

26

hydroperoxide lyase (LOX/HPL). The LOX/AOS pathway results in production of

27

jasmonic acid (JA). The LOX/HPL pathway results in the derivation of some green

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leafy volatiles (GLVs), including C6 aldehydes, and alcohols and their esters. HPL is

29

a key enzyme in the LOX/HPL pathway. In recent years, the LOX/HPL pathway has

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been reported to operate in insect- or pest- induced plant defense.1-4 For instance,

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tobacco plants (Nicotiana tabacum) release herbivore-induced volatiles (such as

32

GLVs) exclusively at night that are highly repellent to female moths (Heliothis

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virescens).2 Moreover, Kessler and Baldwin3 found that volatile emissions from

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Nicotiana attenuata plants during attack by leaf-feeding herbivores increased egg

routes,

mediated

by allene oxide

synthase

3


(LOX/AOS)

or


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predation rates by a generalist predator, that linalool and the complete blend

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decreased lepidopteran oviposition rates, and that a plant, through releasing volatiles,

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could reduce the number of herbivores by more than 90%.

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Fatty acid hydroperoxide lyase (HPL) is an enzyme that cleaves a fatty acid

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hydroperoxide (HPO) derived from the activity of a lipoxygenase (LOX) into two

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carbonyl compounds.5-7 Depending on the substrate specificities, HPLs can be

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divided into three groups: 9-HPL, 13-HPL and 9/13-HPL, which catalyze their

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corresponded

43

12(Z)-octadecadienoic

acid

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15(Z)-octadecatrienoic

acid),

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11(E)-octadecadienoic

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15(Z)-octadecatrienoic acid) and 9- or 13-HPO respectively. 9/13-HPL activity has

47

been reported in the fruits of cucumber (Cucumis sativus), seeds and seedlings of

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soybean (Glycine max) and seedlings of alfalfa (Medicago sativa).8-11 However, only

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13-HPO activity has been reported in watermelon (Citrullus lanatus) seedlings, tea

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(Camellia sinensis) leaves, apple (Malus pumila) fruits, tomato (Lycopersicon

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esculentum) leaves and fruits, green bell pepper (Capsicum annuum) fruits, guava

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(Psidium guajava) Fruit and leaves of potato (Solanum tuberosum).12-18 In pear

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(Pyrus communis) fruits, only 9-HPL has been reported.19 However, two HPL genes

substrates

acid

9-HPO and

(9-HPOD,

9-HPOT,

13-HPO and

9-hydroperoxy-10(E),

(13-HPOD,

13-HPOT,

9-hydroperoxy-10(E),

13-hydroperoxy-9(Z),

13-hydroperoxy-9(Z),

4


12(Z),

11(E),

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were isolated from the leaves of grape berries (Vitis vinifera). VvHPL1 is specific for

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13-HPO with higher activity and VvHPL2 could catalyze the cleavage of both 9- and

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13-HPO.20

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HPLs are widely distributed in plants and produce not only main particular

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volatiles, but also relate to disease-resistance and pest/insect-resistance in plants. The

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first cDNA sequence encoding HPL was cloned from bell peppers in 1996.21 After

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that, HPL sequences from cucumber, guava, arabidopsis, citrus, tomato and other

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plants were reported.17, 22-27 For tea plants, HPL activity was reported previously,13

62

however, cDNA cloning and expression analysis of HPL in tea plants has not been

63

investigated up to now.

64

In our previous studies on the molecular response in tea to feeding by the

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lepidopteran pest Ectropis obliqua, a suppressive subtractive hybridization (SSH)

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library was successfully constructed. A cDNA fragment similar to HPL was found in

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the SSH library, and differentially expressed after Ectropis obliqua feeding. In this

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study, the full length cDNA of HPL in Camellia sinensis (CsHPL) was amplified by

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RACE method, the copy number of CsHPL was determined by genomic DNA gel

70

blotting, the in vitro catalytic function of the gene was validated using recombinant

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proteins expressed in E. coli. and the expression profile of CsHPL was investigated.

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These results would lay an essential foundation for further study of the defense

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function of C6 volatiles in response to insects attack for tea plants.

74

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MATERIALS AND METHODS

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Plant materials and treatments

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Three-year-old clone cuttings of tea plant (Camellia sinensis cv. Shuchazao) were

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cultured with every three tea cuttings grown in one pot (30-cm diameter, 35-cm height)

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and grown under controlled environment at the tea plantation in Anhui Agricultural

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University, Hefei, China.

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For insect feeding treatments, three pots with 9 tea plants were treatment. Tea

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geometrids (Ectropis obliqua) at the 3rd larval stage were placed on tea plants (20

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geometrids per individual tea plant). The first tender mature leaves (from the top)

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were collected after 1/3 of each leaf was consumed by geometrids. To create samples

85

of mechanical damage, tea leaves were sheared by autoclaved scissors to remove

86

amounts of leaf tissue similar to E. obliqua larvae feeding. Tea leaves from

87

non-treated plants were used as controls. The samples were collected at 0, 3, 6, 9 and

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24 h after 1/3 of each leaf was consumed by geometrids or mechanical damage,

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immediately frozen in liquid nitrogen and stored at -80 °C until use. Three

90

replications were carried out individually.

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cDNA cloning

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The cDNA fragment was obtained from our previous studies on genes

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differentially expressed after Ectropis obliqua feeding, which were identified by

95

suppression subtractive hybridization (SSH) (GenBank ID: GW342656). The cDNA

96

was highly similar to HPLs from other plants.

97

The full length cDNA encoding CsHPL was acquired by Rapid Amplification of

98

cDNA Ends PCR (RACE-PCR). Approximately 120 ng of RNA isolated from young

99

tea leaves were used as the template in the RACE-PCR reaction for the cloning of

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CsHPL using the protocol described by the manufacturer (RACE cDNA

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Amplification Kit, Clontech Lab. Inc., USA). 3' RACE-PCR reaction of CsHPL was

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conducted using the primer pair UPM (provided by kit) and GSP3 (5'-

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GACATCTGGCTCGCTCTCCAACTCC-3') at first, and then by nested PCR using

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NUP (provided by kit) and NGSP3 (5'- AAGTTATCCAGAGAGGCGAGACCGA-3').

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5' RACE-PCR amplification was carried out to amplify the 5' end of the CsHPL gene

106

by

107

(5'-GAAACCGCCGTAGGCATTGAAGCCG-3'), followed by using the nested

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primers NUP and NGSP5 (5'-AGTTGGAGAGCGAGCCAGATGTC-3'). The PCR

109

products were ligated into pMD-18T easy vector (Takara, China) after purification.

110

After the 3' and 5' sequences were obtained, the full-length cDNA sequence of C.

first

using

primers

UPM

7


and

GSP5


HPL

was

cloned

by

RT-PCR

reaction

using

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sinensis

112

(5'-AAGCAGAAAGACTCCAAAACC-3')

113

(5'-TCACTTAGCTTTTTCAACGGC-3'), and then sequenced.

and

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primers

FP1 FP2

114

115

Bioinformatic and phylogenetic analyses

116

BLAST search using CsHPL cDNA and protein sequence were performed using

117

BLAST program at the NCBI web site (http://www.ncbi.nih.gov:/BLAST/). The

118

open reading frame (ORF) was found using the ORF Finder. The putative domains

119

were identified using the InterPro database (http://www.ebi.ac.uk/interproscan/).

120

Amino acid sequence alignments were carried out using CLUSTAL X (version 2.0)

121

software. The phylogenetic tree was constructed using MEGA 4.0 package

122

(http://www.megasoftware.net/mega4/mega.html) based on the neighbor-joining

123

method 28 with reliability inferred using the bootstrap method with 1000 replicates.

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125

Southern blot hybridization analysis

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Genomic DNA was isolated from tea leaves using a CTAB extraction procedure.29

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DNA samples (10 µg) were digested with restriction enzymes DraI, SpeI and Bgl II

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(Takara, China) and separated by electrophoresis on 1% agarose gels. DNA was

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blotted onto a Hybond-N+ membrane (Amersham, UK) using a semidry electroblotter.

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DNA blot was hybridized at 42 °C overnight in DIG Easy Hyb buffer (Roche

131

Diagnostics, Germany) with hybridization probes that were prepared by

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5’-oligonucleotide

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5’-ATGGCGAGGCTGGACTCTATG-3’. Probe labeling of DIG (DIG Probe Synthesis Kit,

134

Roche) and membrane washing and detection of target DNA were done as described

135

by the manufacturer.

end

labeling

with

the

following

sequences:

136

137

Preparation of recombinant CsHPL protein in E. coli

138

Plasmid vector pET-32a(+) (Novagen, USA) was used for producing recombinant

139

protein. The open reading frame was amplified by PCR with pfu polymerase (Takara,

140

China) using the cDNA generated from total RNA of C. sinenesis leaves and

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gene-specific primers. A pair of forward primer and reverse primer was designed for

142

amplifying the predicted ORF for CsHPL after analysis by ORF Finder software. 5’-

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CGCGGATCCATGTCGGCAGTGATGGCGA-3’ (forward, Bam HI site underlined),

144

5’-CCCAAGCTTTCACTTAGCTTTTTCAACGGC-3’ (reverse, Hind III site underlined).

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Amplified DNA fragment was introduced into pMD-18T vector and thereafter

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transformed to E. coli DH 5α, resulting in pMD-18T/CsHPL. The plasmid and

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pET-32a(+) were digested with the respective restriction enzymes and the CsHPL

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open reading frame was inserted into pET-32a(+) by ligation to form

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pET-32a(+)/CsHPL.

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After confirming the cloned fragment by sequencing, the construct was

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transformed into the E. coli Rosetta (DE3) pLysS cells (Novagen, Germany). For

152

comparison, only pET-32a(+) vector was also transformed into E. coli cells. Cells

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were grown at 37 °C overnight in LB media containing ampicillin (50 µg ml-1).

154

Following centrifugation, E. coli cells were adjusted to OD600 = 0.3-0.5, and the

155

production of recombinant protein was induced by adding IPTG to a final

156

concentration of 0.2 mM. No IPTG induction samples were treated as controls.

157

Incubation was continued overnight at 16 °C.

158 159

Enzyme activity assay

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Bacterial cells in 1 mL of LB media were collected by centrifugation at 13,000 g

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for 10 min at 4 °C. The pellets were resuspended in 40 ml of PBS buffer (pH 7.0)

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containing PMSF (1 mmol·L-1). After cells were disrupted on ice by ultrasonication,

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both the supernatant and precipitate were collected for SDS-PAGE analysis.

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Meanwhile, the pellets were resuspended in distilled deionized water, boiled for 5

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min, and loaded for SDS-PAGE as total crude proteins. The remaining supernatant

166

samples from the disrupted cells were used for the recombinant CsHPL activity

167

measure. The concentration of recombinant CsHPL was calculated by determining

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the total protein concentration by spectrophotometer and the ratio of target protein in

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total protein by Quantity one software in SDS-PAGE electrophoretogram. The

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substrate (2 µg 13- or 9-HPOT dissolved in EtOH) and the total proteins (40 µL)

171

were mixed with 50 mM potassium phosphate buffer, pH 7.0 to a final volume of 1.0

172

mL. The recombinant CsHPL activity was assayed according to Vick (1991)30 by

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measuring the decrease in absorbance at 234 nm due to disruption of the conjugated

174

diene chromophore of 13- or 9-HPOT.

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After that, 13-HPOT was used as substrate to determine the activity of

176

recombinant protein pET-32a(+)/CsHPL, which were induced by IPTG for 10 h. The

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non-induced pET-32a(+)/CsHPL protein and pET-32a(+) vector were used as control

178

samples. The method for determination of optimum pH and temperature for soluble

179

CsHPL was also carried out using the substrate 13-HPOT.

180

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Product identification

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For the identification of volatiles, 0.5 mL of a solution containing 0.02 mg

183

13-HPOT in 50 mM phosphate buffer, pH 7.0, was prepared. Immediately after

184

starting the reaction by adding 0.5 mL of total proteins, the reaction vessel (1 mL),

185

incubated at 25 °C, was closed with a septum, and a solid phase micro extraction

186

(SPME) fiber, coated with 100 µM polydimethysiloxane (Supelco Inc., Bellefonte,

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USA), was placed above the reaction mixture. Volatiles were collected in the

188

headspace for 30 min, whereupon they were desorbed from the fiber at 200 °C for 1

189

min in the injection port of a GC/MS (Fisons GC 8000 series and Fisons Instruments

190

MD 800 Mass Lab spectrometer). The temperature program was started at the same

191

time as the desorption: it was kept at 40 °C for 2 min then raised by 10 °C/min to

192

200 °C, where it remained for 2 min. The column used was an HP-Innowax, (0.25 µm

193

film thickness; 30 m × 0.32 mm i.d.; Hewlett-Packard, USA).

194

195

Real-time quantitative RT-PCR (qRT-PCR) analysis

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Total RNA was extracted from tea leaves using the RNAprep Pure Plant Kit

197

(Tiangen, China) following the manufacturer’s instruction. cDNA was transcribed

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from 0.5 µg of each total RNA using a PrimeScript RT Reagent Kit (Takara, Japan).

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Real-time quantitative PCR (qRT-PCR) was performed using 2 µl of cDNA product

200

and each primer of 0.4 µM in a 25 µl reaction volume with SYBR Premix Ex Taq™

201

II (Perfect Real Time; Takara) on a Mini-Opticon real-time PCR system (Bio-Rad,

202

USA). The GAPDH (Accession No. GE651107) of tea plants was used as an internal

203

control for transcript normalization. The specific primers used for qRT-PCR analysis

204

were designed on the basis of the 3’ or 5’-untranslated regions (UTR) of individual

205

genes

using

the

Primer

Premier

12


5.0

software

Page 13 of 36


206

(http://www.premierbiosoft.com/primerdesign/). The primer sets for GAPDH were

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5’-TTGGCATCGTTGAGGGTCT-3’

208

5’-CAGTGGGAACACGGAAAGC-3’

209

5’-ATCCCTAACACCGCCATCG-3’

210

5'-CCTTGGAACCAGAAGTAGTC-3’ (reverse). Three replicates of each PCR were

211

run using a program: 95 °C for 30 s, followed by 40 cycles (95 °C for 5 s and 60 °C

212

for 30 s).

(forward), (reverse);

and

for

CsHPL

were

(forward),

213

The amplification efficiencies for all genes tested in this study ranged from 95% ~

214

110%. Data were analyzed according to the threshold cycle (Ct). The relative

215

changes in gene expression were quantified using the 2-∆∆Ct method. The indicated

216

differences are based on Duncan’s multiple range tests using DPS software

217

(www.chinadps.net).

218

219

RESULTS

220

Isolation of cDNA coding for HPL in Camellia sinensis

221

In our previous studies, a cDNA sequence (GenBank ID: GW342656) was

222

isolated from tea plant and BLAST searching against the GenBank database showed

223

high similarity to HPLs. To obtain the complete cDNA sequence corresponding to

224

the cDNA fragment in C. sinensis, 3’ and 5’ RACE were performed. After

13



225

sequencing, a full length HPL of 1662 bp was submitted to GenBank, with accession

226

number HM 440156, and designated CsHPL. The CsHPL cDNA contains a 51 bp

227

5’-untranslated region (UTR) and a 106 bp 3’-UTR, excluding the poly(A) tail.

228

Predicted by the Protean program in DNAstar software, open reading frame (ORF)

229

was predicted. The deduced translation product of CsHPL consisted of 491 amino

230

acids with a relative molecular mass of 54,880 Da and a theoretical isoelectric point

231

of 8.02.

232

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Alignment of HPLs and phylogenetic relationship analysis

234

The deduced amino acid sequence of CsHPL showed the highest homology with a

235

HPL from Psidium guajava (71% identity),17 and 66% identity to a Capsicum

236

annuum HPL.21 CsHPL contains 4 highly conserved domains (Domain A, B, C and

237

D) found in the Cytochrome P450 family (Figure 1). This establishes CsHPL as a

238

new member of the CYB74B subfamily of Cyt P450s. Five putative AUG start

239

codons (encoding Met 1, 5, 8, 9 and 15) in upstream of the predicted coding

240

sequence were present in CsHPL. However, guava (Psidium guajava) has 4 putative

241

start codons,17 of which 2 start codons (encoding Met 1, 9) are the same as that of

242

CsHPL. There are a total of 2 putative AUG start codons in bell pepper (Capsicum

243

annuum), among them the same Met 15 was also found in its protein sequence

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(Figure 1). While many conserved residues were found at the N and C termini, their

245

function and importance remain to be determined.

246

247

Genomic Southern blotting analysis

248

Southern blotting was performed to identify the number of CsHPL-related genes

249

in C. sinensis. Total genomic DNA of tea leaves was digested with restriction

250

enzymes with recognition sites (SpeI, DraI, BglII) not in the CsHPL cDNA. The

251

cDNA of CsHPL was used as hybridization probe. One band was observed in each

252

digestion (Figure 2), suggesting that only one copy of the CsHPL gene exists in tea

253

genomic DNA.

254

255

Expression of CsHPL in E. coli

256

To confirm CsHPL as an HPL-encoding cDNA, recombinant protein was

257

expressed in E. coli and subjected to in vitro functional enzyme assay. The putative

258

ORF of CsHPL in E. coli was induced by different concentrations of IPTG (0.05-1

259

mM) at various temperatures ranging from 15-37 °C. Bacterial cultures induced to

260

express the constructs accumulated high levels of the recombinant protein as

261

determined by SDS-PAGE of bacterial lysates (Figure 3). PAGE showed that a 60

15



262

kD protein was expressed after the recombinant pET-32a(+)/CsHPL was induced by

263

IPTG, which coincided with the molecular weight predicted by DNAStar software.

264

265

Enzyme activity determination and product identification by GC/MS

266

Both the 13- and 9-HPOT substrates were used to determine the activity of

267

recombinant CsHPL. The decrease in absorbance at 234 nm was only found when

268

incubated with 13-HPOT, while the absorbance value was always stable when

269

9-HPOT was employed. The non-induced pET-32a(+)/CsHPL and pET-32a(+) vector

270

E. coli samples were used as controls to evaluate the activity of recombinant CsHPL

271

protein (Figure 4A).

272

After that, 13-HPOT was used as substrate to determine the activity of

273

recombinant CsHPL, which were induced by IPTG for 10 h. The enzymatic activity

274

of CsHPL was measured at different pH (5.0-9.0) at various temperatures ranging

275

from 20 to 40 °C (Figure 4B, C). The optimum pH and temperature were 7.0 and

276

25 °C. The activity of recombinant protein pET-32a(+)/CsHPL was determined with

277

13-HPOT under 25 °C at a pH of 7.0.

278

The reaction with 13-HPOT as substrate and lysates of cells with the recombinant

279

plasmid pET-32a(+)/CsHPL after IPTG induction was determined by Gas

280

Chromatography (Figure 5B), and the chromatogram of control was shown in Figure

16


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281

5A. Gas chromatography/mass spectrometry (GC/MS) was used to identify the

282

metabolite produced upon incubation of 13-HPOT with lysates from bacteria that

283

expressed the recombinant protein pET-32a(+)/CsHPL. The GC profile and mass

284

spectrum identified the volatile product formed as 3(Z)-hexenal (Figure 5C).

285 286

Insect feeding- and mechanical damage-inducible expression of CsHPL in tea leaves

287

The expression levels of CsHPL after Ectropic obliqua feeding and after

288

mechanical damage were quantified by qRT-PCR. Initially after treatment, the

289

transcript levels of CsHPL were similar between insect feeding and mechanical

290

damage (3 h), but increased in the plants attacked by insect such that by 9 h insect

291

feeding induced a 2.9-fold increase in transcript levels compared to 3 h (Figure 6),

292

Transcript levels in both treatments returned to original levels at 24 h after treatment

293

(Figure 6). Mechanical damage induced CsHPL, but not significantly. CsHPL

294

transcript induction reached 1.2 fold at 12 h. A similar trend was found in another tea

295

variety, Camellis sinensis cv. Longjing 43 (data not shown). In this variety, the

296

maximum transcript levels were found at 3 h after insect feeding and mechanical

297

damage, and then gradually dropped back to original values in the following 21

298

hours.

299

17



300

DISCUSSION

301

Fatty acid hydroperoxides produced by 13-lipoxygenase are important

302

intermediates in the oxylipin pathway of fatty acid oxygenation in plants.31 One

303

pathway for 13-HPOT metabolism is initiated by the enzyme fatty acid HPL.

304

Aldehyde products of HPL, together with the corresponding reduced alcohols, play

305

important roles in the odor of fruits, vegetables, and green leaves. Among them, C6

306

aldehydes produced by HPL can act as phytoalexins against protozoa, bacteria and

307

fungi

308

HPL is the precursor of the previously identified “wound signal” known as

309

traumatin.34 In our previous studies on the molecular response in tea plant to

310

Ectropis obliqua feeding, a cDNA fragment from HPL was obtained through

311

suppressive subtractive hybridization (SSH). Herein, the full length HPL in Camellia

312

sinensis (CsHPL) was amplified by RACE. Molecular cloning, transcript expression

313

profile and response, and biochemical activity of CsHPL were investigated.

31, 32

and may be signals for gene regulation.33 The C12 oxo-acid product of

314

CsHPL Southern blotting revealed a simple hybridization pattern (Figure 2),

315

indicating that CsHPL may be present as a single copy in the tea genome. A similar

316

result was reported in almond.35 After comparison with other plants HPLs, the

317

deduced amino acid sequence of CsHPL was found to contain the 4 conserved

318

domains (Domains A, B, C and D), which are the typical structure of Cytochrome

18


Page 18 of 36

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319

P450 family proteins.35 CsHPL carries the highly conserved isoleucine in the I-helix

320

region in Domain A (asterisk, Figure 1), which is replaced by valine in Arabidopsis

321

HPL (Genbank ID: AF087932). The heme-binding cysteines and phenylalanines

322

were also found in the CsHPL Domain D (circle dot in Figure 1). 22, 36 A conserved

323

sequence “LPxRxIPGSYGxPxxGP” indicates that CsHPL is a new member of the

324

CYP74B subfamily of Cyt P450s (underlined in Figure 1).

325

Expression of CsHPL in E. coli illustrated that this cDNA encodes a functional

326

member of the CYP74B subfamily of enzymes. CsHPL catalyzed 13- but not

327

9-hydroperoxides of C18 fatty acid. This feature was also found in HPLs isolated

328

from other plants, for example, bell pepper,

329

GC/MS analysis of the recombinant CsHPL lysate reaction product identified

330

3(Z)-hexenal as the main product.

15

Arabidopsis,

33

and tomato.16,

31

331

Damage inflicted to tea leaves by either geometrid attack or mechanical damage

332

both induced an accumulation of CsHPL (Figure 6). The accumulation of CsHPL

333

transcripts can contribute to understanding its function in plants. After wounding, the

334

enhanced transcription levels of HPL catalyzed the cleavage of polyunsaturated fatty

335

acid hydroperoxides to aldehydes and oxoacids. These aldehydes play an important

336

role in producing the aroma profile in tea leaves. Additionally, they have

337

antimicrobial activity and are thought to be involved in the plant defense response

19



338

against pest and pathogen attack. Further, HPL catabolites may function in a key step

339

of the resistance mechanism of plants against some sucking insect pests or other

340

herbivores.6, 37, 38, 39, 40 It will be interesting to investigate the repression in E. obliqua,

341

since this lepidopteran is a key pest of tea.

342

343

FUNDING SOURCES

344

This study was supported by the National Natural Science Foundation of China

345

(NSFC) projects 30971831 and 31171608, the Changjiang Scholars and Innovative

346

Research Team in University (IRT1101), and the Special Innovative Province

347

Construction in Anhui Province in 2015 (15czs08032).

348

349

References

350

(1) Halitschke, R.; Ziegler, J.; Keinänen, M.; Baldwin, I.T. Silencing of

351

hydroperoxide lyase and allene oxide synthase reveals substrate and defense

352

signaling crosstalk in Nicotiana attenuate. Plant J. 2004, 40, 35—46.

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(2) Moraes, C.M.; Mescher, M.C.; Tumlinson, J.H. Caterpillar-induced nocturnal

354

plant volatiles repel conspecific females. Nature 2001, 410, 577—580.

355

(3) Kessler, A.; Baldwin, I.T. Defensive function of herbivore-induced plant volatile

20


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lyase: a plant cytochrome P450 enzyme involved in wound healing and pest

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Kindl, H., Kajiwara, T.; Feussner I. Biosynthesis of fatty acid derived aldehydes is

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induced upon mechanical wounding and its products show fungicidal activities in

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Shen, X.Q. Isolation, expression and characterizaiton of a hydroperoxide lyase gene

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germinating watermelon seedlings. Plant Physiol. 1976, 57, 80—788.

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Slusarenko, A.; Brash, A.R.; Whitehead, I.M. Purification, molecular cloning, and

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expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit

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(18) Mu, W.; Xue, Q.; Jiang, B.; Hua, Y. Molecular cloning, expression, and

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enzymatic characterization of Solanum tuberosum hydroperoxide lyase. Eur. Food

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lyase from fruits of pear. J. Agric. Food Chem. 1981, 29, 1220—1225.

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(20) Zhu, B.Q.; Xu, X.Q.; Wu, Y.W.; Duan, C.Q.; Pan, Q.H. Isolation and

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characterizaiton of two hydroperoxide lyase genes from grape berries. Mol. Biol. Rep.

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2012, 39, 7443—7455.

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hydroperoxide lyase is a cytochrome P450(CYP74B). FEBS Lett. 1996, 394,

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21—24.

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(22) Matsui K.; Ujita, C.; Fujimoto S.; Wilkinson, J.; Hiatt, B.; Knauf, V.; Kajiwara,

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T.; Feussner, I. Fatty acid 9- and 13- hydroperoxide lyases from cucumber. FEBS

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Lett. 2000, 481, 183—188.

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from fatty acid hydroperoxides: molecular cloning of a hydroperoxide lyase

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(CYP74C) with Specificity for both the 9- and 13- hydroperoxides of linoleic and

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linolenic acids. Arch. Biochem. and Biophys. 2001, 386, 281—289.

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specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase

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from Arabidopsis thaliana. J. Plant Physiol. 2003, 160, 803—809.

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hydroperoxide lyase gene and effect of C6- volatiles on expression of genes of the

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oxylip in metabolism in Citrus. J. Plant Physiol. 2003, 160, 1219—1231.

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secretion of tomato hydroperoxide lyase in Pichia pastoris. Process Biochem. 2005,

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Rochette, L., Husson, F. Characterizati on of purified green bell pepper

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hydroperoxidelyase expressed by Yarrow lipolytica: Radicals detection during

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catalysis. Enzyme and Microb. Technol. 2007, 41, 13—18.

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reconstruction phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406—25.

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(29) Porebski, S.; Bailey, G.; Baum, B.R. Modification of a CTAB DNA extraction

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protocol for plants containing high polysaccharide and Polyphenol components.

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Plant Mol. Biol. Rep. 1997, 15, 8—15.

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(30) Vick, B.A. A spectrophotometric assay for hydroperoxide lyase. Lipids 1991, 26,

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315—320.

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(31) Howe, G.A.; Lee, G.I.; Itoh, A.; et al. Cytochrome P450-dependent metabolism

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of oxylipins in tomato, Cloning and expression of allene oxide synthase and fatty

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acid hydroperoxide lyase. Plant Physiol. 2000, 123, 711—724.

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(32) Blée, E. Phytooxylipins and plant defense reactions. Prog. Lipid Res. 1998, 37,

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33—72.

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(33) Bate, N.J.; Rothstein, S.J. C6-Volatiles derived from the lipoxygenase pathway

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include a subset of defense-related genes. Plant J. 1998, 16, 561—569.

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(34) Zimmerman, D.; Coudron, C.A. Identification of traumatin, a wound hormone,

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as 12-oxo-trans-dodecenoic acid. Plant physiol. 1979, 63, 536—541.

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(35) Mita, G.; Quarta, A.; Fasano, P.; De Paolis A.; Di Sansebastiano G.P.; Perrotta,

447

C., Iannacone, R., Belfield, E.’ Hughes, R.; Tsesmetzis, N.; Casey, R.; Santino, A.

448

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new

449

CYP74 targeted to lipid bodies. J. Exp. Bot. 2005, 56, 2321—2333.

25



450

(36) Noordermeer, M.A.; Dijken, A.J.H.V.; Smeekens, S.C.M.; Veldink, G.A.;

451

Vliegenthart, J.F.G. Characterization of three cloned and expressed 13-hydroperoxide

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lyase isoenzymes from alfalfa with unusual N-terminal sequences and different

453

enzyme kinetics. FEBS Lett. 2000, 267, 2473—2482.

454

(37) Padilla, M.N.; Hernandez, M.L.; Perez, A.G. Isolation, expression, and

455

characterization of a 13-hydroperoxide lyase gene from olive fruit related to the

456

biosynthesis of the main virgin olive oil aroma compounds. J. Agric. Food Chem.

457

2010, 58, 5649—5657.

458

(38) Isabelle, P.; Sandrine, D.; Grit, R.; Jorge, V.; Maria José, R.; Neil, K.

459

Evaluation of the antimicrobial activities of plant oxylipins supports their

460

involvement in defense against pathogens. Plant Physiol. 2005, 139, 1902—1913.

461

(39) Vancanneyt, G.; Sanz, C.; Farmaki, T.; et al. Hydroperoxide lyase depletion in

462

transgenic potato plants leads to an increase in aphid performance. Proc. Natl. Acda.

463

Sci. U.S.A. 2001, 98, 8139—8144.

464

(40) Arimura, G.; Kost, C.; Boland, W. Herbivore-induced, indirect plant defenses.

465

Biochim. Biophys. Acta. 2005, 1734, 91—111.

466

26


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Page 27 of 36


467

Legends for Figures

468

Figure 1. Alignment of amino acid sequences of CsHPL (GenBank accession no.

469

ADO51747) with HPLs from other plant species.

470

Guava, Psidium guajava (PgHPL, AF239670), bell pepper, Capsicum annum

471

(CaHPL, U51674), arabidopsis, Arabidopsis thaliana (AtHPL, AF087932), tomato,

472

Lycopersicon esculentum (LeHPL, AJ239065), tobacco, Nicotiana tabacum (NtHPL,

473

AJ414400) and alfalfa, Medicago sativa (MsHPL, AJ249246) were aligned with

474

CLUSTALW. The A, B, C, D domains for cytochrome P450s are boxed. The

475

conserved sequence LPxRxIPGSYGxPxxGP of CYP74 subfamily of P450 proteins

476

is underlined. The highly conserved isoleucine in Domain A is shown with an

477

asterisk, and the heme-binding cysteines and phenylalanines are shown with dots in

478

Domain D.

479

480

Figure 2. Southern blotting analysis of the CsHPL.

481

Tea genomic DNA was digested with the restriction enzymes SpeI, DraI and BglI

482

and hybridized with a CsHPL probe.

483

484

Figure 3. SDS-PAGE analysis of recombinant CsHPL protein expressed in E.

27



485

coli.

486

CsHPL was cloned into pET-32a(+) vector, and expressed in E. coli (Rosetta (DE3)

487

pLysS). After IPTG-induced protein expression, total cells or cell lysates after

488

sonication were subjected to SDS-PAGE, the gel was stained with Coomassie blue

489

R-250. The lanes M and 1–5 in the graph are as following: M, protein molecular

490

mass marker; 1, total crude protein from uninduced cells were transformed with

491

pET-32a(+) vector alone; 2, total crude protein from induced cells were transformed

492

with pET-32a(+) vector alone; 3, precipitation of total crude protein from induced

493

cells were transformed with pET-32a(+)/CsHPL after sonication; 4, suspension of

494

total crude protein from induced cells were transformed with pET-32a(+)/CsHPL

495

after sonication; 5, total crude protein from uninduced cells were transformed with

496

pET-32a(+)/CsHPL. The lanes 1a, 4a and 4b are the repetition samples of 1 and 4.

497

The bands of pET-32a(+)/CsHPL were pointed out by arrows in the Lanes 3-4.

498

499

Figure 4. Biochemical activity of the recombinant CsHPL.

500

(A): The time-course of the absorbance change with different substrates. A,

501

diamonds, 13-HPOT as a substrate with crude recombinant protein extract induced

502

by IPTG; B, square, using 9-HPOT as a substrate; C, triangle, using 13-HPOT as a

503

substrate with crude recombinant protein extracts without IPTG induction; D, star,

28


Page 28 of 36

Page 29 of 36


504

using 13-HPOT as a substrate with crude protein extracts from E. coli. carrying the

505

empty pET-32a(+) vector induced by IPTG. (B): CsHPL activity on 13-HPOT over

506

temperature range of 20 – 40 °C. (C): CsHPL activity on 13-HPOT over a pH range

507

of 5 – 9. Data represent the means ± SD (n=3). The experiments were repeated 3

508

times individually.

509

510

Figure 5. GC/MS analysis of the volatile products of recombinant CsHPL.

511

(A): Partial GC chromatogram from a reaction with 13-HPOT as substrate and

512

lysates of cells with the recombinant plasmid pET-32a(+)/CsHPL before IPTG

513

induction; (B): Partial GC chromatogram from a reaction with 13-HPOT as substrate

514

and lysates of cells with the recombinant plasmid pET-32a(+)/CsHPL after IPTG

515

induction; (C): GC mass spectrum of the characteristic peak found in (B) was

516

identified to be 3(Z)-hexenal.

517

518

Figure 6. Relative levels of CsHPL expression in tea leaves treated with either

519

Ectropic obliqua attack or mechanical injury.

520

GAPDH was used as internal control. The expression of the genes in non-treated

521

controls was set to 1.0. Data represent the means ± SD (n=3) of three biological

522

samples. The experiments were repeated 3 times with similar results.

29



Page 30 of 36

CsHPL PgHPL CaHPL AtHPL LeHPL NtHPL MsHPL

MSAVMAKMMSISPGMPSPSPSLTPPSPSSISAPVRTIPGGYGWPVLGPISDRLDYFWFQGPETFFRKRIEKHKSTVFRTNLPPTFPFF MARVVMSNMSPAMSSTYPPSLSPPSSPRPTTLPVRTIPGSYGWPLLGPISDRLDYFWFQGPETFFRKRIEKYKSTVFRANVPPCFPFF MIPIMSSAPLSTATPISLPVRKIPGSYGFPLLGPLWDRLDYNWFQKLPDFFSKRVEKYNSTVFRTNVPPCFPFF MLLRTMAATSPRPPPSTSLTSQQPPSPPSQLPLRTMPGSYGWPLVGPLSDRLDYFWFQGPDKFFRTRAEKYKSTVFRTNIPPTFPFF MNPAPLSTPAPVTLPVRSIPGSYGLPLVGPIADRLDYFWFQKPENFFTKRMEKHKSTVFRTNVPPCFPFF MSTLMAKMMSGSTPTNPGSTGTSSPSLTPPPASLPVRTIPGGYGWPLLGPISDRLDYNWFQGPNTFFTKRIEKHKSTVFRTNVPPCFPFF MSLPPPIPPPSLTTPPKARPTELPIRQIPGSYGWPLLGPLSDRLDYFWFQKPENFFRTRMDKYKSTVFRTNIPPTFPFF

88 88 74 87 70 90 79


YGVNPNVVALLDCKSFAHMFNMEIVEKKNVLVGDFMPSVSYTGDLRVCAYLDTSESLHSKVKNFALDILKRSSTIWVPTLSSTLDTMWSS SNVNPNVVVVLDCESFAHLFDMEIVEKSNVLVGDFMPSVKYTGNIRVCAYLDTSEPQHAQVKNFAMDILKRSSKVWESEVISNLDTMWDT LGVNPNVVAVLDVKSFAHLFDMEIVEKANVLVGDFMPSVVYTGDMRVCAYLDTSEPKHTQIKNFSLDILKRSSKTWVPTLVKELDTLFGT GNVNPNIVAVLDVKSFSHLFDMDLVDKRDVLIGDFRPSLGFYGGVCVGVNLDTTEPKHAKIKGFAMETLKRSSKVWLQELRSNLNIFWGT GSVNPNVVAVLDVKSFSHLFDMEIVEKANVLVGDFMPSVVYTGDMRVCAYLDTSEPKHAQIKNFSQDILKRGSKTWVPTLLKELDTMFTT LGVNPNVVAVLDVKSFSHLFDMEIVEKANVLVGDFMPSVKYTGDMRVCAYLDTSEPKHTQIKNFSLDILKRSSKTWVPTLVNELNSMFET TNVNPNIIAVLDCKSFSHLFDMDLVDKRDVLVGDFVPSVEFTGNIRVGVYQDVSEPQHAKAKNFSMNILKQSSSIWVPELISNLDIFLDQ

178 178 164 177 160 180 169


IESSLAMSGSASYLVPIQQFIFSFFTRTLIGADTAASPEIASSGYAMLDIWLALQLLPTVKIGIL.QPLEELFLHSYAYPFFLVSGGYNK IESSLAKDGNASVIFPLQKFLFNFLSKSIIGADPAASPQVAKSGYAMLDRWLALQLLPTINIGVL.QPLVEIFLHSWAYPFALVSGDYNK FESDLSKSKSASLLPALQKFLFNFFSLTFLGADPSASPEIANSGFAYLDAWLAIQLAPTVSIGVL.QPLEEIFVHSFSYPYFLVRGGYEK IESEISKNGAASYIFPLQRCIFSFLCASLAGVDASVSPDIAENGWKTINTWLALQVIPTAKLGVVPQPLEEILLHTWPYPSLLIAGNYKK FEADLSKSNTASLLPALQKFLFNFFSLTILGADPSVSPEIANSGYIFLDSWLAIQLAPTVSIGVL.QPLEEILVHSFAYPFFLVKGNYEK FESDISKSNSASLLPTMQKFLFNFFSLSLLGANPSASPEIANSGYVMLDTWLAIQLAPTVSIGLL.QPLEEIFVHSFNYPFFLVKGSYEK IEATLSKSSSASYFSPLQQFLFTFLSKVLARADPSLDSKIAESGSSMLNKWLAVQLLPTVSVGTI.QPLEEIFLHSFSYPYALVSGDYNN

267 267 253 267 249 269 258


LVKFIEEHGKEVIQRGETEFGLTKHETIHNLLFILGFNAYGGFSIFLPTLLSQLG.TDTTGIQQKLREEVRAKSGS...TLSFDSVKEME LYQFIEKEGREAVERAKAEFGLTHQEAIHNLLFILGFNAFGGFSIFLPTLLSNIL.SDTTGLQDRLRKEVRAKGGP...ALSFASVKEME LIKFVKSEAKEVLTRAQTDFQLTEQEAIHNLLFILGFNAFGGFTIFLPTLLGNLGDEKNAEMQEKLRKEVREKVGTNQENLSFESVKEME LYNFIDENAGDCLRLGQEEFRLTRDEAIQNLLFVLGFNAYGGFSVFLPSLIGRIT.GDNSGLQERIRTEVRRVCGSG.SDLNFKTVNEME LVQFVKNEAKEVLSRAQTEFQLTEQEAIHNLWFILGFNAFGGFSIFLPTLLGNLGDEKNADMQEKLRKEVRDKVGVNPENLSFESVKEME LIQFVKNEAKEVLNRGKSEFGLTEQEAIHNLLFILGFNAFGGFSIFLPTLLGNLGDEKNAELQEKLRNEVREKVGLKTENLSFESVKEME LYNFIKQHGKEVIKSG.TEFGLSEDEAIHNLLFVLGFNSYGGFSIFLPKLIESIA.NGPTGLQEKLRKEAREKGG...STLGFDSLKELE

353 353 343 355 339 359 343

Domain A


*

LVKSFVYETLRLNPPVPLQYARARKDFILSSHDSAYEIKKGELLCGYQTLVMRDSKVFDNPEKFIFDRFTKEKGSELLSYLYWSNGPQTG LVKSVVYETLRLNPPVPFQYARARKDFQLKSHDSVFDVKKGELLCGYQKVVMTDPKVFDEPESFNSDRFVQN..SELLDYLYWSNGPQTG LVQSFVYESLRLSPPVPSQYARARKDFMLSSHDSVYEIKKGELLCGYQPLVMKDPKVFDEPEKFMLERFTKEKGKELLNYLFWSNGPQTG LVKSVVYETLRFNPPVPLQFARARKDFQISSHDAVFEVKKGELLCGYQPLVMRDANVFDEPEEFKPDRYVGETGSELLNYLYWSNGPQTG LVQSFVYETLRLSPPVPSQYARARKDFKLSSHDSVYEIKKGELLRGYQPLVMKDPKVFDEPEKFVLERFTKEKGKELLNYLFWSNGPQTG LVQSFVYETLRLSPPVPSQYARARKDFKLSSHDSVYEIKKGELLCGYQPLVMRDPKVFDDPEKFVLERFTKEKGKELLNYLFWSNGPQTG LINSVVYETLRMNPPVPLQFGRARKDFQLSSYDSAFNVKKGELLCGFQKLIMRDPVVFDEPEQFKPERFTKEKGAELLNYLYWSNGPQTG Domain B


Domain C

SPCESNKQCAAKDYVTLTACLFVAHLYRRYDSITCNSSGAITAVEKA TPTESNKQCAAKDYVTLTACLFVAYMFRRYNSVTGSS.SSITAVEKA SPTESNKQCAAKDAVTLTASLIVAYIFQKYDSVSFSS.GSLTSVKKA TPSASNKQCAAKDIVTLTASLLVADLFLRYDTITGDS.GSIKAVVKA RPTESNKQCAAKDMVTLTASLIVAYIFQKYDSVSFSS.GSLTSVKKA RPTESNKQCAAKDVVTLTASLIVAYIFQRYDSVSFSS.GSLTSVKKA SPTVSNKQCAGKDIVTFTAALIVAHLLRRYDLIKGDG.SSITALRKA ●

●

443 441 433 445 429 449 433

490 487 479 491 475 495 479

Domain D

Figure 1

30


Page 31 of 36


kb

Spe I

Dra I

Bgl II

12.1 6.1 5.1 4.1 3.1

Figure 2

31



M

1a

4a

1

2

4b

3

Page 32 of 36

4

5

94.4 66.2 45.0 35.0 26.0 20.0

15.0

Figure 3

32



Absorbance Units

(A)

0.85

0.80

A B C D

0.75

0.70

0.65 0

1

2

3

4

Time (min)

µ mol HPOTmin -1 mg -1 protein

(B)

0.22 0.20 0.18 0.16 0.14 0.12 20

25

30

35

40

45

Temperature （ ° C）

(C) µ mol HPOTmin -1 mg -1 protein

Page 33 of 36

0.25 0.20

0.15

0.10 0.05 0.00 5

6

7

8

9

10

PH

Figure 4

33



(A)

Page 34 of 36

(x100,000) 3.0 TIC 2.5 2.0 1.5

1.0 0.5

5.1

(B)

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

6.0

6.1

6.2

6.3

Time (min) (x100,000) TIC

6.0 5.0

3-(Z)- hexenal 4.0 3.0 2.0 1.0 0.0 5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

6.0

6.1

6.2

6.3

Time (min)

(C) % 100.0

41 3-(Z)- hexenal

75.0 50.0 25.0

53

49

0.0 40.0

69

55

42

45.0

50.0

55.0

61 60.0

65 65.0

80

70 77 70.0

75.0

80.0

83 86 85.0

98 90.0

95.0

Figure 5

34


100.0

Page 35 of 36


Control Insect feeding Mechanical Damage

Relative Expression

5 4 3 2 1 0 3h

6h

9h

12 h

24 h

Figure 6

35



Table of Contents Graphic 81x77mm (150 x 150 DPI)


Page 36 of 36

Molecular Cloning and Characterization of ... - ACS Publications

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