Molecular characterization of lipoxygenase genes and their expression analysis against biotic and abiotic stresses in Panax ginseng

16 PUBLICATIONS 98 CITATIONS 18 PUBLICATIONS 116 CITATIONS

SEE PROFILE

SEE PROFILE

Johan Sukweenadhi

Deok-Chun Yang

Kyung Hee University Kyung Hee University

19 PUBLICATIONS 46 CITATIONS 538 PUBLICATIONS 3,334 CITATIONS

SEE PROFILE

SEE PROFILE

Molecular characterization of lipoxygenase genes and their expression analysis against biotic and abiotic stresses in Panax ginseng

Kwi-Sik Bae, Shadi Rahimi, Yu-Jin Kim, Balusamy Sri Renuka Devi, Altanzul Khorolragchaa, Johan Sukweenadhi, Jeniffer Silva, et al.

European Journal of Plant Pathology

Published in cooperation with the European Foundation for Plant Pathology

ISSN 0929-1873

Eur J Plant Pathol DOI 10.1007/s10658-015-0847-9

Your article is protected by copyright and all rights are held exclusively by Koninklijke Nederlandse Planteziektenkundige Vereniging. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is

Molecular characterization of lipoxygenase genes and their expression analysis against biotic and abiotic stresses in Panax ginseng

Kwi-Sik Bae & Shadi Rahimi & Yu-Jin Kim & Balusamy Sri Renuka Devi & Altanzul Khorolragchaa & Johan Sukweenadhi & Jeniffer Silva & Davaajargal Myagmarjav & Deok-Chun Yang

Accepted: 18 December 2015 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Abstract Lipoxygenase (LOX) belongs to a family of non-heme-iron-containing fatty acid dioxygenases that are widely distributed in plants and animals. LOX in- volved in the synthesis of jasmonic acid and six-carbon (C6) volatiles which is necessary for plant growth and responses to a wide range of biotic and abiotic stresses. We have isolated and characterized LOX cDNA clones from Panax ginseng Meyer. From their deduced amino acid sequences, two diverse classes of 9-LOX (LOX1, LOX2, and LOX3) and 13-LOX (LOX4, LOX5) are defined in P. ginseng. A GenBank Blast X search revealed that the deduced amino acid of PgLOXs share a high degree of homology with LOX from other plants and mammals especially in three distinct motifs; motif1 har- boring iron binding regions, motif2 and motif3.

Chloroplast localization was predicted for PgLOX5. PgLOXs displayed organ-specific expression, highly expressed in aerial parts of the plant such as 3-year old flower, stem and leaf tissues. PgLOXs mRNAs were elevated strongly by bacterial infection. Expression of PgLOXs was differentially induced in ginseng not only by mechanical damage and methyl jasmonate but also after exposure to withholding water. Ginseng 13-LOXs positively respond to wounding that may involve in pro- duction of C6 volatiles and jasmonic acid at the wounded sites. However, the higher expression of PgLOX3 by water deficit and 82 % of the nucleotide sequence identity with the EST from severe drought-stressed leaves of Populus (CU229089.1) at +6371 bp downstream of PgLOX3 genomic DNA structure can suggest drought tolerance role for PgLOX3. Ginseng LOX genes have different expression pattern which may suggest different specific function against various environmental stresses.

Keywords Abiotic stress . Biotic stress . Gene expression . Jasmonic acid . Lipoxygenase . Panax ginseng

Abbreviations AOC

allene oxide cyclase AOS

allene oxide synthase EST

expressed sequence tag

HPL hydroperoxide lyase 13-HPOD (13S)-hydroperoxyoctadecadienoic acid 9-HPOD

(9S)-hydroperoxyoctadecadienoic acid IBA

indole-3-butyric acid

DOI 10.1007/s10658-015-0847-9

Kwi-Sik Bae and Shadi Rahimi contributed equally to this work. Electronic supplementary material The online version of this

article (doi:10.1007/s10658-015-0847-9) contains supplementary material, which is available to authorized users.

D.<C. Yang Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea

K.<S. Bae : S. Rahimi ( *) : Y.<J. Kim :

B. S. R. Devi :

A. Khorolragchaa : J. Sukweenadhi : J. Silva :

D. Myagmarjav : D.<C. Yang (*)

Graduate School of Biotechnology and Ginseng Bank, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea e-mail: shadirahimi@khu.ac.kr e-mail: dcyang@khu.ac.kr

JA jasmonic acid oxylipins. 13-HPOD can be further metabolized into LOX

lipoxygenase jasmonates in plants. 13-LOX and 9-LOX can be dif- MJ

methyl jasmonate ferentiated by their gene expression patterns and subcel- OPDA

(9S,13S)-12-oxo phytodienoic acid lular localizations (Shen et al. 2014 ). LOX form gene Pst

Pseudomonas syringae pv tomato strain families ranging from six in Arabidopsis to at least 18 DC3000

genes in potato which differently localized in microsom- PUFAs

polyunsaturated fatty acids al membranes (Feussner and Kindl 1994 ), plasma mem- RT-PCR

reverse transcriptase-polymerase chain branes (Nellen et al. 1995 ), and lipid bodies (Feussner reaction.

and Kindl 1992 ) of cucumber cotyledons, cytosol and vacuole of soybean leaves (Grayburn et al. 1991 ; Stephenson et al. 1998 ), envelope of leaf chloroplasts in spinach, barley, tomato, potato or Arabidopsis (Blée

Introduction and Joyard 1996 ; Feussner et al. 1995 ; Heitz et al. 1997 ; Royo et al. 1996 ). Different LOXs may have different

Panax ginseng Meyer, a perennial herbaceous plant function in plants. There are different four major fates from Araliaceae family, is one of the most commonly

for the LOX-derived hydro (pero) xy PUFAs in plants utilized medicinal plants. Due to long cultivation period

(Feussner and Wasternack 2002 ). First is hydroperoxide of ginseng, various ecological stresses might affect the

isomerase pathway which converts fatty acid hydroper- plant and this condition makes this highly valued me-

oxides to epoxy- or dihydrodiol polyenoic fatty acids, dicinal plant more susceptible to environmental stresses.

second is AOS pathway which forms unstable allene However, plants activate distinct defense responses

oxides further metabolized to OPDA by an AOC. Third which are mediated by a complex hormonal network

is the HPL pathway oxidatively cleaves the hydrocarbon to adapt with adverse conditions during their growth and

backbone of fatty acid hydroperoxides and forms short development. Jasmonates belong to the oxygenated

chain aldehydes (C6- or C9-). Fourth one is the DES compounds known as oxylipins are essential signaling

pathway forms divinyl ethers such as colneleic acid or molecules in response to environmental changes as

colnelenic acid. Ketodiene-forming pathway, epoxy al- well as growth and development. Oxylipin biosynthesis

cohol synthase pathway and reductase pathway are oth- is initiated by the function of LOX enzyme

er reactions for hydro (pero) xy PUFAs metabolisms (EC1.13.11.12) which is ubiquitously occurring in plant

which is less studied.

and mammals and catalyzed hydroperoxidation of poly- Since JA will be rapidly accumulated in response unsaturated fatty acids. Interestingly, LOX-derived

to wounding or herbivore attack to confer direct oxylipins are involved in wound healing and defense

defenses at the site of attack and systemic defense processes in plants, while in mammals they are involved

signaling (Howe and Jander 2008 ), the characteriza- in inflammation, asthma and heart disease (Andreou and

tion of LOX as the critical protein catalyzed the Feussner 2009 ).

initial step of JA pathway will help us to understand Two different isomer products of 13-HPOD and 9-

the plant defense mechanism in response to ecolog- HPOD are synthesized by introduction of molecular

ical stresses. Despite of the importance of LOX, oxygen either at carbon atom 9 or at carbon atom 13

there is a dearth of genetic evidence for the function of hydrocarbon backbone by the function of plant LOX

of this defensive gene in the ginseng plant. On the which can justify their role as 13-LOX and 9-LOX,

one hand, weeds compete with ginseng to absorb respectively (Wasternack 2007 ). LOX function can

water and nutrients and on the other hand, due to change from 13–to 9-LOX by a change in histidine

the long cultivation period, ginseng is frequently residue of substrate binding site (Hornung et al. 1999 ).

exposed to bacterial infection and herbivores. In 9-LOXs are a subfamily of proteins which share high

these stressful conditions, ginseng root growth will amino-acid sequence identity (~60 %) to one another,

be affected and it can decrease the worth of this but 13-LOXs are sharing only ~35 % sequence identity

economically important plant. among themselves (Andreou and Feussner 2009 ; Park et

In this report, we attempt to identify the five LOX al. 2013 ). The discrete pathways are proposed for 13-

genes in P. ginseng and understand the biological sig- LOX and 9-LOX which can lead to numerous types of

nificance of these genes in the initial step of plant nificance of these genes in the initial step of plant

and root) were collected from Ginseng Bank, Korea. genes at different organs was examined. The present study examined the phylogenetic relationship of ginseng

Stress treatment

LOXs and that of other organisms, to contribute to the understanding of the possible role of ginseng LOXs.

To investigate the response of the PgLOX genes to MJ elicitor, 100 μM MJ, was applied to 30-day-old subcultured adventitious roots, and harvested at 6, 12,

Materials and methods

24, 48, 72 h after treatment. The P. ginseng seeds used in the present study were obtained from the Korean Identification of the LOX genes from the P. ginseng

Ginseng Resource Bank, South Korea. The 4- week- EST database

old ginseng seedlings germinated in in vitro condition on solidified MS media (Duchefa biochem, Netherland)

To identify genes from the EST library, we used the EST with 3 % sucrose, supplemented with gibberellic acid library constructed from P. ginseng (Sathiyamoorthy et

(10 mg L −1 ) at 25 °C under a 16 h photoperiod and used al. 2009 ). Homologous sequences of LOX ESTs were

for the treatment studies. To induce drought stress, searched against the GenBank databases using a

plants were exposed to dehydration by withholding BLASTX algorithm. We identified and selected the

water. Ginseng plants were inoculated with the virulent LOX genes based on open reading frames encoding

Pseudomonas syringae pv tomato (Pst) strain DC3000. the specific protein via the BlastX program (NCBI

The bacteria were grown at 28 °C in King’s B medium BlastX program). Clustal X with default gap penalties

containing rifampicin (50 mg L −1 ). To study bacterial was used to perform multiple alignments of LOXs iso-

infection, bacterial suspensions (5 × 10 6 cfu mL −1 ) were lated from P. ginseng and previously registered in other

infiltrated into the ginseng leaves using a syringe. At species. A phylogenetic tree was constructed by the

appropriate time points, three independent leaves infil- neighbor-joining method, and the reliability of each

trated with Pst were harvested. For wounding experi- node was established by bootstrap methods using

ments, leaves were pricked with a needle (Hwang and MEGA6 software. The protein properties were estimat-

Hwang 2010 ). For stress treatments, the plantlets were

ed using ProtParam (Gasteiger et al. 2005 ) and the subjected to treatment for 6, 12, 24, 48 and 72 h. Control hydropathy value was calculated by the method de-

plants were grown at 25 °C under a 16 h photoperiod. scribed by Kyte and Doolittle ( 1982 ). Identification of conserved motifs within LOXs was accomplished with

RNA extraction and quantitative RT-PCR analysis MEME (Bailey et al. 2009 ). Chloroplast localization was inspected by ChloroP (Emanuelsson et al. 1999 )

Total RNA was extracted from adventitious roots of P. and proposed signal peptide is predicted from PSORT

ginseng using RNeasy mini kit (Qiagen, Valencia, CA, (K. Nakai, Tokyo University, http://psort.ims.u-tokyo.

USA). For RT-PCR, 200 ng of total RNA was used as a ac.jp/ ). Other database also used to analyze the PgLOX

template for reverse transcription using oligo (dT) 15 genes, such as MotifScan ( http://myhits.isbsib.ch/cgi-

primer (0.2 mM) and AMV Reverse Transcriptase (10 bin/motif_scan ), HMMTOP ( http://www.enzim.hu/

U μL −1 ) (INTRON Biotechnology, Inc., South Korea) hmmtop ), SOPMA ( http://npsa-pbil.ibcp.fr/cgi-bin/

according to the manufacturer’s instructions. Real-time npsa_automat.pl?page=/NPSA/npsa_server.html ).

quantitative PCR was performed using 100 ng of cDNA in a 10-μL reaction volume using SYBR® Green

Plant materials and growth conditions Sensimix Plus Master Mix (Quantace, Watford, England). Gene-specific primers listed in Table 1 , were P. ginseng adventitious roots were collected from

used to perform quantitative RT-PCR. The thermal cy- Ginseng Bank, Kyung Hee University and grown for

cler conditions recommended by the manufacturer were

1 month in liquid Murashige & Skoog medium used as follows: 10 min at 95 °C, followed 40 cycles of (Murashige and Skoog 1962 ) supplemented with

95 °C for 10 s, 58 °C for 10 s, and 72 °C 20 s. The

2 mg L −1 IBA at 25 °C. The roots were maintained by fluorescent product was detected at the last step of each regular subculture every 4 weeks. Different organs of 3-

cycle. Relative quantity of the PgLOX genes

Table 1 List of gene-specific primer sequences used for qRT- possibly mediate interaction with lipids or membrane PCR analysis

bound proteins and the C-terminus domain of LOX Gene

Primers (5’–3’) superfamily possessed by all the previously cloned plant and indicated that PgLOX1, PgLOX2, PgLOX3,

PgLOX1 F: ATCAAGAGAGGGATGGCAGTTGA PgLOX4, and PgLOX5 belonged to the LOX family. R: ATGGCCCTTTTCTCGGACTTCT

Red underlined sequence showed the proposed signal PgLOX2

peptide in Fig. 1 predicted by IPSORT program, show- PgLOX3

F: TACTGATCGAGGACTACCCATATGCA R: CATTTTAGGCCATGCTTCAT

F: ATGTTGCCATTTCTGCTCAAGTTC ing that PgLOX5 had a transit peptide at its N-terminal

R: CAGATATCCAACCACGACAAGCA which may suggest chloroplast localization. Multiple PgLOX4

F: CAAGTCATTCTTACCATCTCAAAC sequence alignment revealed that the N-terminal se- R: CCTTTAGTGTGAACGATGTCTCCT

quences differ among the LOXs in different plants, PgLOX5

F: TATGTGCCTAATCGTCCCACTCTCAT including five PgLOXs (Fig. 1 ). Plant LOX enzymes R: CCTGAACCAAGTACTCTTCATCTGG

contain iron atom in their active site which coordinated Pgβ-actin

F: GTGATCTTACAGATAGCTTGATGA by 5 five amino acids including three histidines, one R: AGAGAAGCTAAGATTGATCCTCC

asparagine and the carboxy group of the carboxy- terminal isoleucine. Two iron-binding regions were shown in green boxes in motif1. In addition, three

transcription level was performed using Rotor-Gene conserved motifs were found by MEME analysis in all 6000 real-time rotary analyzer (Corbett Life Science,

plant LOX isozymes and mammals (Fig. 2b ) Three Sydney, Australia), and calculated using the compara-

conserved motif sequences predicted by MEME are tive cycle threshold (CT) method according to the man-

shown in Online Resource 1 .

ufacturers’ instructions for normalizing data. A consti- It was revealed that the deduced amino acid of tutively expressed β-actin gene was used as internal

PgLOX1, PgLOX2 and PgLOX3 share a high degree reference. Three independent experiments were per-

of sequence homology with 9-LOX proteins encoded by formed. The primer efficiencies were determined ac-

Actinidia deliciosa (ABF60002.1) (identity 74 % & cording to the method by Livak and Schmittgen

similarity 99 %), Camellia sinensis (ABW75772.2) ( 2001 ) to validate the ΔΔCt method. The observed

(identity 79 % & similarity 100 %), Capsicum annuum slopes were close to zero, indicating that the efficiencies

(ABF19103.2) (identity 78 % & similarity 98 %), re- of the gene and the internal control β -actin were equal.

spectively. However, PgLOX4 and PgLOX5 share a high degree of sequence homology with chloroplastic 13-LOX proteins encoded by Camellia sinensis

Results (ADO51752.1) (identity 66 % &similarity 100 %) and Theobroma cacao (XP_007045017.1) (identity 78 % &

Isolation and characterization of PgLOX genes similarity 90 %), respectively. Clustal X and the MEGA6 Program were used for the construction of

As the part of a genomic project to identify genes of the phylogenetic tree based on LOX amino acid sequences medicinal plant P. ginseng, a cDNA library consisting

(Fig. 2 ). Moreover, the phylogenetic relationship is about 20,000 cDNAs were previously constructed. Five

shown among LOXs from P. ginseng and Arabidopsis LOXs, designated PgLOX1, PgLOX2, PgLOX3,

thaliana (Online Resource 2 ). Transmembrane helices PgLOX4, and PgLOX5 were isolated. Moreover, full

of integral membrane proteins and localization of the N- genomic DNA sequence of PgLOXs was analyzed using

terminus were predicted by HMMTOP and mentioned the genomic DNA sequence retrieved from ginseng

in Table 2 . The hydrophobicity profile of the estimated genome database ( http://im-crop.snu.ac.kr/new/index.

9- and 13-LOX proteins is shown in Fig. 3a, b . N- php ). The corresponding characteristics of each

terminal peptides differ from each other but more hy-

drophilic amino acid composition similar to other LOXs Fig. 1a GenBank Blast search ( http://www.ncbi.nlm.

PgLOXs are indicated in Table 2 . As it was shown in

were found in PgLOXs.

nih.gov/BLAST/ ) resulting in highly conserved N- Secondary structure analysis and molecular model- terminus domain of PLAT/LH2 which is found in a

ing for PgLOXs were conducted by SOPMA (Geourjon variety of membrane or lipid associated proteins to

and Deleage 1995 ) (Table 3 ). The secondary structure

Table 2 Characteristics of ginseng LOXs Protein

Length a Molecular mass a pI a Predicted

c localization localization b Transmembrane helices of N terminus c

PgLOX1 815

Out PgLOX2

Out PgLOX3

Out PgLOX4

Out PgLOX5

Out a Length (number of amino acid residues), molecular mass (kDa), and isoelectric point (pl) of PgLOX proteins deduced from the open reading frames (Table 2 ). Values for predicted mature proteins are indicated in brackets

b Transit peptides of PgLOX proteins were predicted by the IPSORT c Transmembrane helices of integral membrane proteins and localization of the N-terminus were predicted by HMMTOP

analysis revealed that PgLOX1 consists of 312 α-heli- Expression of PgLOXs in response to MJ elicitor, ces, 74 β-turns joined by 128 extended strands, and 301

wounding and bacterial infection randomcoils; PgLOX4 consists of 268 α-helices, 73 β- turns joined by 143 extended strands, and 278

To determine the expression of the PgLOX genes in randomcoils. PgLOX5 consists of 335 α-helices, 77

response to MJ, we performed quantitative RT-PCR β -turns joined by 144 extended strands, and 352

analysis of LOXs in ginseng adventitious roots treated randomcoils which is highly similar to the secondary

with 100 μM MJ (Fig. 5 ). MJ elicitor upregulated structure of At13-LOX6 from A. thaliana, includes 346

PgLOX5 6 h after treatment however, PgLOX4 and α -helices, 62 β-turns joined by 140 extended strands,

PgLOX3 were highly responsive 12 h and 72 h after and 369 random coils; and At9-LOX from A. thaliana,

MJ treatment. PgLOX1, PgLOX2 and PgLOX3 expres- made up of 335 α-helices, 70 β-turns joined by 149

sion were not responsive to mechanical damage. extended strands, and 332 randomcoils. Similar second-

PgLOX4 was highly upregulated 72 h after wounding ary structure were found in PgLOX2 and PgLOX3

treatment however, PgLOX5 was showed higher tran- which consists of 297 α-helices, 90 β-turns joined by

script level at 48 h and then it was declined after 72 h. 146 extended strands, and 327 randomcoils and 294 α-

To recognize pathogen can induce the transcriptome helices, 95 β-turns joined by 141 extended strands, and

profile of LOXs, we infected 30-day old ginseng seed- 338 randomcoils, respectively.

lings with Pseudomonas syringe pv tomato strain DC3000 suspension and checked the transcription pat- tern of these genes using quantitative RT-PCR analysis.

Distribution of LOX genes transcript in different organs The expression level of all PgLOXs showed similar pattern with maximum transcription at 48 h after bacte-

To determine the expression of PgLOX genes at differ- rial infection followed by decreased expression 72 h ent various organs of ginseng, we performed quantita-

after bacterial treatment. The mRNA level of PgLOX2 tive RT-PCR expression analysis of PgLOX genes in 3-

was induced by pathogen attack however it was lower

year-old leaf, stem, flower, and root samples (Fig. 4 ).

than other PgLOXs.

The results indicate that these LOX enzymes are tissue- regulated showing PgLOX1, PgLOX4, and PgLOX5 transcripts were highly detected in flower bud whereas

Expression of PgLOXs in response to drought stress relatively higher levels of PgLOX2 and PgLOX3 tran- scripts were observed in leaf and stem, respectively.

To investigate the possible role of LOXs in response to Three-year-old root tissue contain lowest level of

drought stress, plants were exposed to dehydration by PgLOX1, PgLOX4 and PgLOX5 transcripts when

withholding water. The expression patterns of PgLOXs PgLOX3 transcriptome level appears to be same as that

at different time points after treatments were analyzed in leaf root tissue.

using real-time PCR. Among all PgLOXs, only PgLOX3 using real-time PCR. Among all PgLOXs, only PgLOX3

Fig. 1 Multiple amino acid sequence alignment of PgLOXs with „

file of PgLOX3 highly increased at 24 h of post treatment those of proteins encoded by Actinidia deliciosa (ABF60002.1), with drought and after that declined at 48 h and 72 h after Camellia sinensis (ABW75772.2), Capsicum annuum (ABF19103.2), Camellia sinensis (ADO51752.1) and Theobroma

cacao (XP_007045017.1). A hyphen was inserted within the genomic DNA sequence of PgLOX3 has a length of

treatment compared to the control (Fig. 5 ). The full

amino acid sequence to denote a gap. Shadow box indicates well 2,604 bp encoding 906 amino acids. This gene contains

conserved residues, * represents a conserved amino acid, and: nine exons interrupted by the eight introns (Online represents a very similar amino acid. Red underlined sequences are the proposed signal peptide predicted by IPSORT program. Resource 3A ). By carefully checking the genomic

The arrow indicates the conserved domains. Three conserved DNA structure of PgLOX3, a nucleotide sequence with

motifs obtained by MEME analysis. Two iron-binding regions 438 bp size was found at +6,371 − +6,808 bp down-

are shown in green boxes

stream of PgLOX3 genomic DNA structure that shares the highest nucleotide sequence identity (82 %) with the EST from severe drought-stressed leaves of Populus

the chloroplast. However, there was no homology (CU229089.1). A GenBank Blast X search revealed that

among the N-terminals of the LOX. Surprisingly, N- the deduced amino acid of this sequence share a high

terminal chloroplast transit peptide was not identified in degree of homology with Photosystem I reaction center

13-LOX ginseng LOX4. However, there are some ex- subunit D-2 (PsaD2) from other plants (Online Resource

amples for occurrence of enzymes within or at the 3B ) which its Arabidopsis homolog (NP_171812.1) is

plastid without transit peptide. Despite the lack of a highly expressed 24 h after drought treatment.

putative chloroplast signal peptide in AOS sequences of barely, immunocytochemical analysis showed their chloroplast localization (Maucher et al. 2000 ). Those

Discussion chloroplast proteins without transit peptide are located in outer envelope membrane of chloroplast which

In our experimental study, for the first time we have imported through the transit peptide-independent path isolated and characterized the LOX genes in P. ginseng.

(Keegstra and Cline 1999 ).

These sequences were named PgLOX due to their high The temporal expression analysis from different or- homology with the LOX sequences of other plants. It

gans demonstrated that PgLOX mRNAs were ubiqui- was revealed that the deduced amino acid of PgLOX1,

tously expressed in all organs that we examined though PgLOX2 and PgLOX3 shares a high degree of sequence

an expression level of LOXs is low in root tissue. LOXs homology with 9-LOX proteins however, that of

were strongly expressed in aerial parts of the plant, PgLOX4 and PgLOX5 shares a high degree of sequence

especially in 3-year old flower, stem and leaf tissues. homology with chloroplastic 13-LOX proteins.

Since, JA is involved in regulation of pollen maturation, Phylogenic tree analysis of LOXs from different species

anther dehiscence, and flower opening (Scott et Al. suggested that the ginseng LOXs are classified into two

2004 ); we can suggest that higher expression of LOXs groups of 9-LOX and 13-LOX with respect to their

in ginseng flower will provide evidence for their possi- positional specificity of fatty acid oxygenation. Plant

ble role in producing JA during flower development. LOX genes encoded around 94–100 kDa protein se-

Ginseng plants have evolved effective mechanisms quences which contain two different domains. PLAT

to deal with the ever-changing environment during their domain around 25–30 kDa at the N-terminus consists

growth and development. Understanding these mecha- of b-barrel domain (Corbin et al. 2007 ) and possibly

nisms is helpful to improve stress tolerance in ginseng mediates interaction with lipids or membrane bound

plant. In order to determine the molecular mechanisms proteins. There are around 55–65 kDa domain at the

of ginseng plant adapted to biotic and abiotic challenges C-terminus which conatins a-helices and includes cata-

over long years of life cycle, transcript profiling of lytic site of enzyme (Andreou, and Feussner 2009 ).

PgLOXs in response to wounding, MJ supplementation, LOXs belong to a family of non-heme-iron-containing

pathogen attack and drought was studied by quantitative fatty acid dioxygenases that are widely distributed in

real time-PCR analysis. LOX expression in plants is plants and animals. Two iron binding regions were

regulated in response to stresses. In our current study, identified in PgLOX enzymes. PgLOX5 contain transit

ginseng LOXs transcript level exhibited different results. peptide at its N-terminal which directed the enzyme to

LOX1, LOX2 and LOX3 classified as the 9-LOXs, were

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 10 20 30 40 50 60 70 80 90 100 110 120 130 PgLOX1 PgLOX2

---------------- ---------------- M M S R K V ID P I EEE D TS K R M K F E V ---- H E N M K IK GTVVLMKKN L F E L NN -------------------------- IL AS V R DR F D E I LG H KVSLQLISA A N I D ------------ 72 PgLOX3

------------- M G Y F PP F H K C I N S LE E A M V I C T G G E K N R ------------- Q H K S I E N ------- G E E R N R KK K IK MR GTVVLMKKN G R VVLMKKN V V LD LD F F N S D D -------------------------- -------------------------- F L A N S AS S I F LDR LDR V V H H E E L F LG LG K R G KVSLQLISA VSLQLIS S V V P N C G D Q A P G E Q N --------- --------- 75 66

9-LOX Ca9-LOX AdLOX2 ---------------- --------------------- M L E K L L N V V C G R N H D TT DE NN C NNN H T N G KK VR GTVVLMKKN V LD L T D -------------------------- V G AS F LDR V H E V F G K G VSLQLISA D H A E P G N --------- 79

PgLOX4 Cs9-LOX ---------------- M L H R V VE MD ---------------------------------------------------------------------------------------------------------------------------------- P G I I K T G DE N D N ------------- M N KK T K H E V ---- G G N E D I KK KK IQ IK GTVVLMKKN GTVVLMKK K F V LD LD S F N N D D -------------------------- -------------------------- F L N T AS AS V I LDR T DR F V D H E E L I LG LG Q N KVSLQLISA KVSLQLISA V V N N A G D D P L T E V K --------- K -------- 70 67

13-LOX TcLOX PgLOX5 ML

CsLOX ML T

I ML N T L A K S N Q P P T L

--- K

H P P Q S S -- F K H S N D T TS F V Q S I G L P S T A I R R P V Q TS - V A G R F ST RR L T QQQ I P R V K G K Q G S V T A V I S S G D S K S SST V ED S K Q LV S L NNNNNNNN I P L R W H R K P P S R F L L N S G G T V P A S G P -------- SSS LL R L K LV P Q G F F S C W G T N Q I R K T D K R H G R L V V R R A C V V I P S ST -- I D K D A K I A A L TT E S A T A E KK Q S TTS ---- V K SS A VV V E Q K N V D G G S S L V K A L SS T V GG S IL L Y S --- --- S G S S S S G V IE K E V V R K A A V V V I T T I I R R K K K K I M K K E E K K I M S T E E K K I I E E N D Q Q W E S F V ------

117 N L G L S H G LD D I A DL LG K S I Q L E L V SA E L D ------------- W E L F I N G I G Q G 115 113

PLAT domain 250 260

PgLOX2 PgLOX1 ------------ ------------ G E LR GKLG E PA F LE D W S TKIT P VT A S EA S F N VTFDW D E E N I GVPGAF L I K N N H S N EFY -- LKT L TL E D V PGHG - QLY F I CNSWVYPA K Y YK R D RVFF T NQTY I PS Q TPAPL R Y YR E EEL 187 Ca9-LOX PgLOX3

------------ ------------ G S VR IR GKLG GKLGK S A PA A Y Y LE LE D N W W I V TKI T T IT S P S VT L N A A G G ES DA A G F F D NI VTFDW TFDW D E E E S D - - I GVPGAFII R N L HH S EFY -- L R T L TL A D V PG Q C G K GKLGKPA FM E N W VS T L T S IS A G DA T F N VTFDW D E S - M M G GVPGAF F PGAFII V I R K N N Y H HH HH G SQ Q FY L Y -- -- L LKT R T V V TL E D F PGHG - - H RV I HF HF V I CNSWVYPA CNSWVYPA N H K R Y YK TT Y N D RVFF RVFF A S N NQTYLPS -- YLP H S E TPAPL TP E PL L RE E YR YR E K EEL EDL

9-LOX AdLOX2

Cs9-LOX ------------ ------------ E G LK LR GKLGKPA GKLGKPA N Y LE LE D D W W I D TKIT T T IT P A LT LT A A G P G DS V T A F Y D N VTFDW VTF E W E D E E E E - - I I GVPGAFII GVPGAFII R K N N F S HH HH S N EFY EFY -- -- LK LKT SL V TL TL V L E E D D V V PGHG PGHG - - RV QL HF HF V V CNSWVYPA CNSWVYPA S H R C YK YK Y K N D RVFF RVFF A T NQTYLP N K TYLPS TE N TP TPAPL E PL R R C P YR YR E E EEL EEL

PgLOX4 ------------------------------------- D H V PGHG - RV HF V CNSWVYPA K N YK T D RVFF S NQTYL L S E TPAPL I E YR KQ EL 184 181

13-LOX PgLOX5 TcLOX ------------- ILI Q LI S EE I D P V T T K S G K S M E SFV R G W L P K P T N H S N - EI I V E E Y Y D AA C N K F F T T V V P P DE H D - - F F G G E Y PGA I GA I V LV II T Q N N F D H H D S S K E EF M E F T -- FF L M N E SI V E I V L H D G G L F S T K E G S P G -- K QI I F II F N P C G HT E SWV W IH H T S K R Y N A D N N P P D E S K R R I I I FF F K T N N E KSF A YLPS LPS Q Q TPA TP K G G L L V KD Q L R R R R KD EDL DL 228 92

CsLOX ------------- P N K T S G G L K S E V K E E T TS - V IK R G G W Y L A P H R K K P S S E Q E H K S D H - - I EV L K E Y Y AA E C D N : F F VI T * I P P E S D G - - F Y G G E K PGA I GA * V **.:: * * V LVE L I T N N E L HH H G K K EF E M Y H -- -- LK L L E I V I H G F EE G P -- I F F P A N T W IH S R N D N P E S R I L F R NQ A H LPS Q TP P G L KD L R R EDL 237

N I :. V F H G . F P P G G - P V :. D V T CNSWV .:*: . A S K F D S P H K .*::* * R I FF T N KS YLPS .: .** Q TP D G * L K R L *.::* R E EDL 226

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 270 280 290 300 310 320 330 340 350 360 370 380 390 PgLOX1 PgLOX2

D VN T LRG LRGNG T G T G K LEEWDRVYDYD T YNDL S D PDK G I KH V RP I LGGS S D YPYPRRGRTGRPP A KT - D A R N ESRLPL Y E SL K IYVPRDERFSH L K M SD L A AYA V K L I F Q F LVPE F E A M F N R - T F D EFD K L E DILK 315 PgLOX3 Ca9-LOX

IN L S LRGNG T S G G K LEEWDRVYDY A Y YNDLG D PDK D P KY ARPVLGGS S EYPYPRRGRTGRP S TK E A DP K T ESRLPL VM SL N IYVPRDERF G H L K L SDF L AYALKS IV Q F L L PE F E A L C D S - T P D EFD T F Q D V LK 309

Cs9-LOX VN LRG D G K G K LEEWDRVYDY A Y YNDLG D PDK G S KY V ARP RP I I LGGS LGGS T S EYPYPRRGRTGRPPTKT EYPYPRRGRTGRPP A E T - - DP DP R E H SESRL ESR I PL A L F LM K SL S F N S N IYVPRDERF IYVPRDERFSH T H L V K K L F SD SDF L V L AY AYALKS G LKS A L V G Q Q F V L I VPE I PE I VE V A A Q L F F D D S K - - T T P I S D EFD EFD K S F F E E DIL D V LK Q 312 PgLOX4 320 PgLOX5

9-LOX AdLOX2 L T LRGNG LRG S G N S G G V LKEWDRVYDY LKEWDRVYDY A Y YNDLG S P K ---- M LEEWDRVYDYD A F I YNDLG YNDL S F E P PDK E K G G P P KH EY EH V ARPVLGGS RPVLGGS K K EYPYPRRGRTGR EYPYPRRGRT S R T R PTKT A TKT - - D D H L N N SES T ESRLPL Q LP P L L G S - - L L D IYVPRDERFSH I K F SDF L AYALKS L G Q I I VPE IK A I F D K - T P H EFD T F Q D V L N 310

L L V K RGNG T G Q R E A G DR I YDYD V YNDLG E PD E DE N L ARPVLGG - KDH PYPRR C RTGRP H TK -- D T L SESR E ---- V D K F IYVPRDERF YVPRDE T FS G H D L V K K E M SDF T S F L T L AYALKS K G L L G IL VV Q Q F R LVP LVPE L L LK G A K L Q C M ET D K - G K T P E N P EFD G F P Y S F F Q T A DILK I D K

13-LOX TcLOX

M L L SESR C SESR ESR I ----- -- E K P S H ST P V V YVPRDE YVPRDE A A F EE I K Q N T F S A G R LK ALL H N L I P S I A FS A F EE D V I K K E Q L N T T F F S S A A K G A R LK V Y ALL S VL H H A N LVP LVP S S LE I A T A A T S L L S S S S - - S S D D I I P F A C F T IV DI D - T E L G P F F T P Y C F F S T DI A I D D D K K 362 353 ** *

E L SI SV RGNG RGNG K K C G E E R R K K A L H H DR DR I I YDY YDYD S P V YNDLG YNDLG S PDK S E D LV RPVLG N - Q E R PYPRR N C LRGNG Q G E R K T YE R I YDYD V YND I G N D PDK PD SS DE P D TS L K ARPVLGG RPVLGG - - KQH E E R PYPRR PYPRR C C RTGRP R RTGRPPTKT S GRPPTKT - R S KT - - DP DP DP

I -- E K P H P V YVPRDE T

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 400 410 Lipoxygenase iron-binding catalytic domain 420 430 440 450 460 470 480 490 500 510 520

PgLOX1 PgLOX2 LYEGGIKL LYEGGIKLP S - - D E G G P P I L L L D E R N IQE IRE R H IP IP L S E EML L LKEL R EL L L R R S T DGE DG A P G L A A F K KFP Y P L M P P Q Q VIK VIK E - DKSAWRT DKSAWRT -- -- DEEFAREMLAG D K EFAREMLAG M I NPV NPV V S I I - C RL Y L - - Q K FPP - SKL N P Q V I F C D KN C Q S Y R I K S Y E - K Y Q L A I N - I AI Q T N R L Y ILD H 438 PgLOX3 Ca9-LOX

9-LOX H 434

LYEGGIKLP LYEGGIKLP - A E D H HH K L L T N K K - - F LRQ RE S C IP IP W W EMLKEL EMLKELVR I R S S DGE DGE PP QL LKFP LKFP M L P P D D VIK VIK - A DKSAWRT D R SAWRT -- -- DEEFAREMLAGVNPV DEEF G REMLAGVNPV S I I I R R RL RL H - QEFPP QEFPP QEFPP G T SKLDPKVYGN SKLDPKVYGN Q Q T G SSIT SSIT R R E N H D I IE D N N N N - R V L K E GLT GLT VD IE - - Q E V A VKQ VKK N NKLFILD R LFILD

H 432

AdLOX2 LYE K GIK K V P N F P L L E S IR N Q IP L Q T LKEL L R T DGE Q P F R FP T P Q VIK E DKSAWRT -- DEEFAREMLAG I NPV V I C RL - QEFPP A N SKL SKLDPKVYGN N P Q VY N N Q E A T SS SSIT K T K R E E C H IE IE K K N N - - L L E D GLT GLT VD ID - - E E A AI L NN E Y KKLF NKLFILD T LD Y 437 H 443 Cs9-LOX PgLOX4

L I F YEGGIKLP N E G V D L G - - E E E G S P ED L L D T L K L IKE N - L N S IP P R L V EMLKELVR V K STT V V T G DGE II KQI G Y LKFP L R F E T M P P Q ELL VIK D S E DK DK F T AW AWRT M E -- T K DEEFAREMLAGV DEEF G R Q T LAG L D NP PV C S I I I R S RL - QEFPP R S T LDPK L YGN Q N SSIT ED H I K N N - L D G F T IE PgLOX5 - LY S E G F L L R - D EE H K E E V G N Q F L R KLM N - Q V S T V GE R F LK Y E I P A I IK - D R F AW L R -- D N EFAR Q A LAGVNPV N I E L L V L - - K QE EFP W P IL L K SKLDP S D LDP T V N S YG YG S P P T E E S S A A IT IT K R E E L VV IE E QQ QE I L F H - G M M E N V T E T E V V E Q Q E AI K N N R LFILD H 433

13-LOX TcLOX LY

AI AI S E Q N KKLFILD KKLFILD Y 477 Y 341 CsLOX

L F N S E D G G V V N I L LP K - - D P DE L S Q K R N G E L F LKD G NN L L F L I P G R L N V M M K -- K - F Q V V T L D S V A G E E QKL G L L LK R Y F E E T I P P A A I L I F R R D R ::. *. F : :: * :: E R *: :* DK F S AW W F L R R -- -- DEEF D N EFAR Q T LAGVNPV N I E I L - K EFP IL SKLDP A I YG PP E S T IT K E L IE QE L H G M S V D -- K AI EE K R LFILD F *:**.*: ***::* 485 S R Q T LAG L NP Y S * I Q L V : :::* - K E W P L K * *:* SKLDPK I YG PP E S . A IT : K E L IE .: RE I R - G F M T L E V .:. ::*: **. 127 A LQK KKLF M LD Y 471

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 530 540 550 560 570 580 590 600 610 620 630 Motif2 640 650

Iron-binding region

PgLOX2 PgLOX1

Y GA E A I V S S T K VYTP VYTP A T E E E R G G V A K EG G S T IWQLAKAYVAVNDSGYHQLI IWQLAKAYVAVND Q G I HQLISHWL C HWL N H THA THA A S IEPFIIATNRQLS IEPF V IATNRQLSVL I L -- -- HPIYKLLHPHFRDTMN HPI H KLLHPHFRDTMN 559 PgLOX3 565 Ca9-LOX

HD HD A T L LMPYLRRIN V PYLR G IN - - L T T A S A TK TK I T YA YA T T RTLLFL RTLLFL KNN QN DGTLKPL GTLKP V AIELSLPHP V IELS V PHP E E GD GD E Q N

HD HD A A LMPYLRRIN LMPYLRRIN - - T T T T T K TK TK T T YASRTLLFL YASRTLLFL QD QDN DG GTLKPLAIELSLPHP K LKPLAIELSLPH A Q Q GD GD K K Y H GA GA T T S S L Q V V F F TP TP ADE A EDG I EG SV WQLAKAY A AVNDSGYHQLISHWL N THA V IEPF V IATNRQLSVL -- 9-LOX HPIYKLLHPHFRDTMN AdLOX2 HD I LMPYLRRIN - S T S TK I YA T RTLLFL KN DGTLKPLAIE M SLPHP E D D K L G E V S E VYTP A E H G G A V EG EG T TV IWQLAKAYVA WQLAKAY A AVNDSGYHQLISHWL I NDSGYHQLISHWL N Y THA THA V V IEPF IEPFIIATNRQLSVL V IATNRQLSVL -- -- HPI HPI HR F KLL Q PHFRDTM Y 570 561

PgLOX4 Cs9-LOX HD A LMPY V RRIN - A T S TK I YA T RTLLFL Q K DGTLKPLAIELSLPHP N GD Q F GA I S K VYTP S E Q G V EG SV WQLAKAYVAVNDSGYHQLISHWL N THA A IEPF V T ATNRQLSVL -- HPI H KLLHPHFRDTMN LLHPHFRDTMN 564 560

13-LOX TcLOX PgLOX5 HD HD M L L L L L P PY FIKKM V N I V R N S D L L P E A G T T L Y G SRTL F FL T P HD I M L L P FI RR M N N L P G R K K K A A YASRT YASRT V I F F F F Y Y N S R K T T G G N H I L LKP R P I I I AIEL IELSLP V R P P P G TSSS K G K P P -- -- Q Q W N K K R Q V VY F T R P H G G T N D D A STTS TT H W W IW L W K K LAKA Q AKA H H V V C L S A ND H DSGYHQL A G I HQL V VN SHW HWL R - R THA TH C A C M IEP EP Y Y A IIAT IA S NRQL H RQLS G A S M M -- E T HPIYKLLHPH HPIY R LLHPHFR M R Y Y TM T L E E 468 603

CsLOX ** *:*::. :. HD L L L PY V N KV R E S K G T T L Y G SRT I F FL T P DGTL M L T P I AIELSLP P T P SSS -- R N K Y VYT Y G H D A TT H W IW K LAKA H V C S ND A G V HQL VN . *.:**::* HWL . * *: **: M PLAIEL T R P * P V D G .. K P -- Q W . *: K Q V F TP T W D . A T G C W L :*: ***:. W R LAKA H A L A H :* * ***: ** DSGYHQL V SHWL T R TH THA C C T M **. EP V EP Y IIAT **: Y IIA *::***. : S NRQLS H RQLS S A M M -- -- HPIYKLLHPH HPIY R LLHPHFR M R Y Y TM T L E ***.:**:**:* *: E 597 611 192

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 660 670 680 690 700 710 720 730 740 Motif3 750 760 770 780

PgLOX2 PgLOX1 -- -- INALARQ INA F ARQ I T LIN LIN A G GG GG L V LE LE K K TVFPG TVFP E Q KYAME Y S ME -- -- MSS MS A IV N M YKN YK E - - WVF W G F P T EQALPADLIKR EQALPADLIKR - R GMAVED GMAVED S S K N -- -- S S PHG K HGL V R C LLIEDYP LLIEDYPYA F AVDGLEIWSAIKTWV A DGLEIWSAIKTWV KD EE YC YC T N FYY FYY S K TD T NEM EI V V Q QN E D D N Y ELQSWW K E V R EK GHGD 688 PgLOX3 Ca9-LOX

-- -- INALARQ I LIN S GG V LE R TVFP A K FS ME -- MSS F V YKN - WVF T EQ S LPADL L I R R GMAV P D S S -- Q PHGLKLLIEDYPYAVDGLEIW F AI E N WV N E YC S FYY P TD ELIE C D S ELQ ELQSWW L WW K L E E L L R R N EQ E GHGD GHGD 681 684

9-LOX

Cs9-LOX AdLOX2 -- -- INALARQ V I LIN A GG I LE INA R NALARQ F ARQ I T LIN LIN G A GG D G I L LE LE K R TVFPGKYAME TVFP TVFP A S KYAME KYAME -- -- MSS IV Y R N - WVF T EQ G LPADL L KR - G -- V MS L S AV AVV A YKN YK G - - WVF WVF P P EQALPADLIKR EQALPADLIKR - - G G V V AV AVED AV P D L S S K -- Q P Y GLKLLIEDYPYAVDGLEIW E AI E A WV DD YC S FYY S TD DMIR K G D D N -- -- A S PHG PHG IR VR LLI LLIEDYPYAVDGLEIWSAIKTWV Q D C PYAVDGL K IWSAI E TWV QE ED YC YC N S S FYY YY K K N TD D DIIQ EM V K E E D D D S ELQSWW K E V R DE GHGD

692 L L ELQSWW PgLOX4 ELQSWW K N E E L L R R EE EK GHGD GHGD 682 686

CsLOX -- INALAR E A LIN A GG II E T C F S PGKY SI E -- L SS V A Y D Q L W R F D L QALPADLI S R R R R - GMAVED GMAVED P P S S -- -- V V P P N G GLKL GLKL V V IEDYPYA IEDYPYA A A DGL DGL L L IWSAIK E L V E S Y V D H YY S E V N S V T S D V ELQ A WW T E IK N K GH F D :**:**: *:*..* :* 727 . * ::::* :.: * ** :.***:*: * *: . : - GMAVED Q T -- A PHGL R L T IEDYP F A N DGL L IWSAIK V W D AIK E Q WV WV T E S D Y Y V V E K H H YY F Y T Q D E L A S N F S IQS V T S D D K V ELQ E I Q A A WW WW T D E E IK I R N T R V G GHGD N Y D *: * *:* *:* *** :* :*: 733 * .* :* .: * *:: ** *:: *: * 720

13-LOX TcLOX PgLOX5 -- -- T E INALARQ INALARQ N S LIN LIN G GG T I E T INALARQ S S L V N G G GG GG VI II E E A A C S F F F S A PGKYAME PGKY S ME T -- E F I SS C S S V PGKYAME -- L SS AA A G A YKN Y D K M L W R F Y QE ES - W W R R F F D D D M G LPA E M LI E E ALPADLI S ALPADLI R R R - GM E T A E ED P E A P Y GLKL A IEDYPYA S DGL I L W D S IKTWV T D Y V S H YY P E A S L V ES D E EL N A WW T E I R T V GHGD 597

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 790 Motif1 800 810 820 830 840 850 860 870 880 890 900 910

PgLOX1 KKDEPWWPKMQTR PgLOX3 PgLOX2 KKDEPWWP KK N E A WWPKMQTR K D ELI ELI D D S S CT M CTTIIWVASALHAAVNFGQYPY MQTR G EL V L S CT I I IIWVASALHAAVNFGQYPYAG IIW I G SALHAAVNFGQYPYAGYLPNRPT G GYLPNRP A L M SRR S S R -- -- F FMPEP I PEP G S -- -- TPEY TPEY D EL E SNPD K AFLKT I T S Q M L SV LG I S -- L V EILSRHS A DE VF LG -- QRD SS EWT A D V E P R E A Ca9-LOX 810 L KDEPWWP Q MQTR A EL V Q A CT I IIW T ASALHAAVNFGQYPYAGYLPNRPT F LPNRPT V V SRR SRR -- -- FMPEP FMPEP G G -- -- TPEY T A EY A A D EL EL KAD E SNPD PD S L V AFLKT FLKT I I T T A S Q Q F L P P TL TL LG LG V IA S -- -- LIEILSRHS LIEILSRHS S T DE DE V L YLG YLG -- -- QRD QRD T TA P EWT EWT K D I E P L N A 803 9-LOX AdLOX2 KKDEPWWPKMQT H K EL V E T CTTIIWVASALHAAVNFGQYPY G GYLPNRP A M SRR -- F I PE R D -- TP D Y A EL EL E E SNP SNPD E L K AFLKT A Y LKT V I T T P A Q Q F M L Q SI TL LG LG V I S S -- -- LIEILSRH LIEILSRHS T A S DE DE I VF YLG LG -- -- QRD QRD N T P P EWT EWT T S S D D I H R Q P A R L QS 814 E A 806

Cs9-LOX KK H EPWWPKMQTR R ELI D S CT I V IWVASALHAAVNFGQYPYAGYLPNRPT L SRR -- FMPEP G -- TPEY E EF K S S PD K AFLKT I T A Q L Q TL LG V S -- LIEILSRHS S DE V YLG -- QRD SAD WT T D D K D E E P A L L E K A A 804 808 PgLOX5 PgLOX4

KKDEPWWP K RN EPWWPK V LK L D T T KED P QD L LI S G G IL IL TTIIWVAS TT M IW I AS G G LHAAVNFGQY Q HAA I NFGQYP D YAGY F G GY V F PNRPT PNRPT L IT M RR R T -- N M F E T I P P T Q ED ED R -- S K D E PEY S WK EK S F F L L S Y R NP P Q E G H E S FL I L L S C SL F P P T T Q Q L F Q Q A A T T L K V V MA M E TS -- V VLD Q D T A LS LS N T HS HS P P DE DE E E YL Y I V G Q -- V A HH K P L Q E P S H A W W I ED R D E H P E VI V VK R A L A 13-LOX 725 851

CsLOX TcLOX KKDEPWWP K RN EPWWPK :.*.*** : * V L LK A T T P KED QD L SS IL TT M IW I AS G Q HAA I NFGQYP F G GY V PNRPT L M R K -- L I P Q E T -- D P DFEK :* F LI G IL TT * :** M IWV T *. *:*:***** :.*:.****:: S G H H S AVNFGQY M YAGY F PNRPT IA R T -- K MP * T ED P T DE .: E WK C : F I I N H NP K P E Q V H A T FL L L M S SL C F P P S TKL Q I Q Q A A T T K K V V MA MA -- -- V VLDV Q D T LS LS N T HS HS P P DE DE E E YLG YLG Q -- M K N Q D L M H E S A S S W W I I N E N D H P E II V L K A K M A 844 *: 857 * ...:: : : : : ** *:.** :: .* :

....|....|....|....|....|....|....|....|....|....|....|....|.... 920 930 940 950 960 970 PgLOX2 PgLOX1

FERFG ----------------------------------------------------------- PgLOX3

FERFG F G RFG T E KL KL V V -- -- EIE EIE K N K R I I M L A N RNNDE M NNDE K R W H KNR

F Q RF H D R L V -- DV E K K I V E RNND S R W KNR KNR N L V GP GP V V N K M VPY PY T M LL LL Y Y P P N ----- TS D Y T T R S N Q S G G L I TGKGIPNSVSI TGKGIPNSVSI

9-LOX AdLOX2

Ca9-LOX

GP V K VPY

FERFG F G RFG E K KL KL T G -- -- EIE EIE E D M R I I I T R E M M NNDE NNDE N K L L R R NR NR T V GP GP A K M PY T M LL LL Y F P P N ----- A S G D T N S S E E V S G G L L TGKGIPNSVSI T V KGIPNSVSI

Cs9-LOX

PgLOX5 PgLOX4

F FE Q K V F F S S A G K KL M E E T -- K EIE DL E R E I I I V DE K R RN RN A K DE D N H K LKNR L R NR C A G G A A G G V K V VPY VPY T K LL LL F K P P ----- ----- T F S S E D P G G G V L TGKGIPNSVSI TG Q G V PNSVSI

3-LOX

CsLOX TcLOX

FE FERF K F N S G A KL KL T G -- -- E EIE L E G E V T I I N D K G RN RN V K D D K I R N LKNR LKNR S C G G A A G I I P P PY PY E E LL LL L P ----- S S A P G V G TG V VPY E LL K L P P ----- ----- F S S S E G P P G G V V TGKG TG R R GIPNS GIPNS I I SI

not responsive to wounding which is in consistent with downregulation of cucumber LOX in response to the previous report by Yang et al. ( 2012 ) showed the

wounding. Meanwhile, LOX4 and LOX5 which belong

100

GhLOX[AAK50778.4] 89 TcLOX1[XP_007030817.1] MdLOX[AGK82779.1]

43 100

PdLOX[CAB94852.1] VvLOX1[AGU28274.1]

66 50 CsLOX[ABW75772.2] 60 PgLOX2

93 OeLOX[ACG56281.1]

9-LOX

99 Nb9-LOX[AGL96414.1]

Plant

76 100 CaLOX1[ACO57136.1] Sl9-LOXA[NP_001234856.1]

100

St9-LOX[NP_001275357.1] AdLOX2[ABF60002.1]

100

100

PgLOX1 99 AaLOX[BAH57745.1]

84 DkLOX[AHX56188.1] 64 PgLOX3

OeLOX1[ACD43483.1] 90 MdLOX[AGK82783.1]

100

Gs9-LOX[KHN02707.1]

42 MtLOX[XP_003592410.1] 69 Ga9-LOX5[KHG03761.1]

100

52 At9-LOX[NP_188879.2] 47 TcLOX[XP_007045017.1] 93 MdLOX6[NP_001281012.1]

89 MnLOX6[XP_010103197.1]

MtLOX[XP_003637315.1]

99 At13-LOX6[NP_176923.1] Gs13-LOX3-1[KHN39622.1]

100

OeLOX2[ACD43484.1] CaLOX2[AFU51541.1]

98 100

100

St13-LOX3-1[NP_001275115.1]

53 PgLOX4 Mn13-LOX2-1[XP_010091997.1]

100

OeLOX2[ACD43485.1] 50 60 CsLOX[ADO51752.1]

13-LOX

71 27 NaLOX[AAP83137.1] MdLOX[AGK82787.1]

100

SlLOX[NP_001234259.1] 68 Mn13-LOX2-1[XP_010087356.1]

50 78 MdLOX[AGK82791.1] TcLOX1[XP_007014894.1] 40 PdLOX2[AAZ57445.1]

41 Prokaryote PdLOX1[AAZ57444.1]

PaVRFPA09LOX[EVT86795.1]

Animal

MmLOX[AAC79681.1]

100

HsLOX[AAA36183.1]

Fungi

Af293LOX[XP_746844.]

0.2

Fig. 2 Sequence homology analysis of PgLOXs with other LOX shown in red and green boxes, respectively. b Organization of genes. a Phylogenetic relationships in LOX from various organ-

putative motifs in LOXs identified by MEME. Numbered color isms including plant species. Database accession numbers are

boxes represent different putative motifs Motifs 1, 2, and 3 are indicated in brackets. Distances between each clone and group

indicated by the mint (First), blue (middle), and red (last) boxes, are calculated with Clustal X program. 9-LOX and 13-LOX are

respectively. Motif sizes are indicated at the bottom of the figure

to 13-LOXs, were highly induced in mechanically dam- JA pathway and its first step catalyzed by allene aged ginseng seedlings and it can be correlated to their

oxide synthase. The other one is biosynthesized specific function in production of related oxylipins

C6 volatiles which initiated by oxidative cleavage which can help to relieve the tension. Three po-

of hydroperoxides through the action of HPL. The tential fates for LOX-derived hydroperoxides were

third pathway is the C5 compounds synthesis and identified by Shen et al. ( 2014 ). One of them is

in that LOX oxygenate hydroperoxides.

Fig. 3 Superimposed hydrophilicity profiles for 9- and 13-PgLOXs and homologous plant LOXs. Hydrophobic domains are indicated by positive numbers; hydrophilic domains are above the line and hydrophilic domains are below the line

LOX-derived C6 volatiles accumulated in wounded Rothstein 1998 ). Also, JA is an essential signaling plants and acted as the signal molecule to induce defense

oxylipin produced highly in wounded plants. There are and repair mechanisms on the damaged site (Bate and

six LOX genes in tomato (Solanum lycopersicum).

Table 3 Secondary structure predictions for PgLOXs and homol- accumulation when elicited. These reports can provide ogous plant LOXs

support for our study that the higher induction of PgLOX Protein

α -Helix β-Turn Extended strand Random coil genes can affect endogenous JA level and it can conse- quently change the secondary metabolite content by

PgLOX1 312

regulating expression level of secondary metabolite bio- PgLOX2

synthetic genes (Reviewed by Rahimi et al. 2015a ; PgLOX3

Rahimi et al. 2015b ; Devi Balusamy et al. 2015 ). At9-LOX

C6 volatiles and JA are the essential molecules for PgLOX4

plant response to biotic stress, as it was already shown PgLOX5

LOX derived (E)-2-hexenal released from infected At13-LOX6 346

leaves with bacteria strongly inhibit Pseudomonas syringae growth (Croft et al. 1993 ) and the aerial treat- ment of Arabidopsis with that induced expression of

TomloxA, TomloxB, and TomloxE encoded 9-LOXs and defensive genes (Bate and Rothstein 1998 ). Higher their functions are not related to C6 biosynthesis

mRNA levels of PgLOXs during bacterial infection (Griffiths et al. 1999 ; Chen et al. 2004 ). However, other

can suggest their role in response to biotic stress. LOX three chloroplast-localized LOXs of TomloxC, TomloxD

pathway can produce hydroperoxy, hydroxy and keto and TomloxF involved in generation of JA and C6

fatty acids upon infection by pathogen especially during volatiles (Chen et al. 2004 ).

the hypersensitive reaction via LOX action. It was sug- Among all PgLOXs, only PgLOX3, PgLOX4 and

gested that these LOX pathway products can regulate PgLOX5 transcriptions were affected by 100 μM MJ

pathogenesis-related and oxidative-stress-related genes in the adventitious roots of P. ginseng as suggesting that

as well as of cell death (Weber 2002 ). LOX-derived α- those LOXs were elicitor-responsive. In most plants,

DOX pathway was induced by Pseudomonas syringae wound-responsive LOXs are induced by JA (Porta et

infection in Arabidopsis and α-DOX reaction products al. 1999 ) and PgLOX5 which was early responsive to

can be suggested to protect plant tissues from oxidative mechanical damage, up-regulated earlier than PgLOX4