LOX genes in blast fungus (Magnaporthe grisea) resistance in rice

ORIGINAL PAPER

LOX genes in blast fungus (Magnaporthe grisea) resistancein rice

Soma S. Marla & V. K. Singh

Received: 17 November 2011 /Revised: 22 January 2012 /Accepted: 7 February 2012 /Published online: 28 February 2012# Springer-Verlag 2012

Abstract Plant Lipoxygenases (LOX) are known to playmajor role in plant immunity by providing front-line defenseagainst pathogen-induced injury. To verify this, we isolateda full-length OsLOX3 gene and also 12 OsLOX cDNAclones from Oryza sativa indica (cultivar Pusa Basmati 1).We have examined the role played by LOXs in plant devel-opment and during attack by blast pathogen Magnaporthegrisea. Gene expression, promoter region analysis, and bio-chemical and protein structure analysis of isolated OsLOX3revealed significant homology with LOX super family. Pro-tein sequence comparison of OsLOXs revealed high levelsof homology when compared with japonica rice (upto100%) and Arabidopsis (up to 64%). Isolated LOX3 geneand 12 OsLOX cDNAs contained the catalytic LOXdomains much required for oxygen binding and synthesisof oxylipins. Amino acid composition, protein secondary

structure, and promoter region analysis (with abundance ofmotifs CGTCA and TGACG) support the role of OsLOX3gene in providing resistance to diseases in rice plants.OsLOX3 gene expression analysis of root, shoot, flag leaf,and developing and mature seed revealed organ specificpatterns during rice plant development and gave evidenceto association between tissue location and physiologicalroles played by individual OsLOXs. Increased defense ac-tivity of oxylipins was observed as demonstrated by PCRamplification of OsLOX3 gene and upon inoculation withvirulent strains of M. grisea and ectopic application ofmethyl jasmonate in the injured leaf tissue in adult riceplants.

Keywords Lipoxygenase . Rice . Blast disease . Defenseresponse .Methyl jasmonate . Expression

Introduction

Rice is a major cereal of more than half the World’s popula-tion, primarily in Asia. Blast disease caused byMagnaporthegrisea is a major concern for rice farmers that significantlyreduces grain yields. Development of varieties with en-hanced and durable resistance against the invading path-ogen is a viable strategy considered by rice breeders. Ithas been reported that lipid turnover in plants influencesvarious physiological functions during development andenvironmental stress (Ohta et al. 1992; Feussner andWasternack 2002; Liavonchanka and Feussner 2006). Inlipid alteration, formation of oxidized fatty acids cata-lyzed by Lipoxygenases (LOXs) is a major reaction.LOXs catalyze addition of molecular oxygen to poly-unsaturated fatty acids (PUFAs) containing (Z,Z)-1,4-pentadine system to produce unsaturated fatty acid

Electronic supplementary material The online version of this article(doi:10.1007/s10142-012-0268-1) contains supplementary material,which is available to authorized users.

S. S. Marla (*)Molecular Biology and Genetic Engineering,G.B. Pant University of Agriculture and Technology,Pantnagar, Indiae-mail: [email protected]

V. K. SinghBioinformatics Centre,Banaras Hindu University,Varanasi, Uttar Pradesh, India

Present Address:S. S. MarlaNational Genomic Resources and Bioinformatics,National Bureau of Plant Genetic Resources, ICAR,Pusa campus, Inderpuri,New Delhi, India

Funct Integr Genomics (2012) 12:265–275DOI 10.1007/s10142-012-0268-1

http://dx.doi.org/10.1007/s10142-012-0268-1

hydroperoxide. LOXs (linoleate/oxygen oxidoreductase,EC 1.13.11.12) belong to non-heme iron-containing fattyacid dioxygenase family of genes, abundantly found inplants and animals (Brash 1999; Feussner and Wasternack2002). In plants, linolenic (LA) and linoleic acids (LeA) arethe most common substrates for LOXs (Siedeow 1991). Jas-monic acid is a popular oxylipin formed from oxygenation offatty acids LA and LeA.

In plants, LOXs are multi-functional enzymes catalyzingvarious types of reactions of which dioxygenation of lipidsubstrates is of prime importance. Functional activity ofenzymes is based on location in plant tissue and LOXs areclassified based on their positional specificity of active site ofLA oxygenation. LA can be oxygenated either at carbon atom9 (9-LOX) or at C-13 (13-LOX) of the hydrocarbon backboneforming the (9S)-hydroperoxy and the (13S)-hydroperoxyderivatives of PUFA (Feussner and Wasternack 2002; Vello-sillo et al. 2007). Hydroperoxides are highly reactive andrapidly degrade in to precursors for synthesis of jasmonicacid, methyl jasmonate (MeJ), conjugated dienoic acids andseveral volatile aldehydes. LOXs are known to play importantroles in signal transduction and induction of defense responseswhen challenged with biotic and abiotic stresses (Kolomites etal. 2000; Eckardt 2008; Gao et al. 2011). Diverse roles playedby different LOXs during growth, development, and environ-mental stress can be explained by the versatile catalytic activ-ities of LOXs and region specificity of various oxylipins inplant tissues (Liavonchanka and Feussner 2006; Porta et al.1999; Gao et al. 2011). Composition of catalytic products(oxylipins) viz. jasmonates, anti-fungal and antimicrobialcompounds such as leaf aldehydes, divinyl ethers, and a seriesof volatile leaf alcohols is frequently altered in different planttissues during development and environmental stresses(Kachroo and Kachroo 2009). The multifunctional nature ofLOXs in plants and their region-specific roles can be betterexplained by generation of data employing comparative mo-lecular and structural studies.

Rapid developments in structural and functional genomicsled to isolation of LOX genes and their characterization inanimals and plants (Lütteke et al. 2003; Liavonchanka andFeussner 2006; Porta and Rocha-Sosa 2002). Transcription ofmembers of this gene family is under tight developmentalcontrol, and often more than one member is active at a specificdevelopmental stage, resulting in multiple LOX isoforms.LOX genes isolated from various plant species show differen-tial organ specific expression patterns (Griffiths et al. 1999;Kolomites et al. 2000). Reported LOX gene functions arediverse, ranging from defense responses against stress, suchas wounding caused by pathogens and herbivores (Farmer andRyan 1990; Melan et al. 1993; Porta et al. 1999), insects(Wang et al. 2008),M. grisea infection (Ohta et al. 1992; Penget al. 1994), and Asperagillus in groundnut seeds (Burow et al.2000) and aroma in rice (Suzuki et al. 1999; Kenta et al 2008).

Also, other LOX functions include response to abiotic stressessuch as drought (Bell and Mullet 1993) and salinity (He et al.2002). However, studies explaining definite functions ofLOXs in cereals, especially in rice are limited.

In the present work, we report isolation and characteriza-tion of a full-length OsLOX3 gene and OsLOX cDNAs fromdifferent organs of rice employing sequence, expression,and structural studies. Analysis of gene expression afterphysical wounding and ectopic application of MeJ con-firmed instantaneous induction of MeJ and expression ofOsLOX3 gene. Furthermore, we report presence of proteinstructural elements and over-represented motifs known to beassociated with plant disease and stress responses that in-clude synthesis of MeJ in the promoter region of OsLOX3gene in Pusa Basmati 1 which might contribute to theresistance of blast disease.

Materials and methods

Plant material and disease inoculation

Seeds of Oryza sativa Pusa Basmati 1 (indica cultivar),Nipponbare (japonica cultivar) obtained from the Depart-ment of Plant Breeding, Agricultural College, G.B. PantAgricultural University (India) were soaked on wet filterpaper in the dark followed by surface sterilization with0.1% HgCl2. The etiolated 25-day-old seedlings were har-vested and used for isolation of genomic DNA. Sampleswere collected from green-house-grown Pusa Basmati 1 riceplants from different organs during various growth stages(root and shoot at 25 days, shoot at 60 days, flag leaf, milkygrain, grain hardening, and mature seed) for construction ofcDNA libraries. Disease scores were recorded for blastdisease between 1 and 14 days after inoculation using a 0–5 scale. Five fungal isolates of rice blast disease (Vii-1084-2, Vii-1085, Vii-1087-1, Vii-1089, and Vii-1092-1) wereprovided by Plant Pathology laboratory of G.B. Pant Uni-versity of Agriculture and Technology, Pantnagar, India.Specially designed small chambers maintaining congenialday and night temperatures, humidity, and light conditionsin a green house were used for disease inoculation. GenomicDNA extracted (Murray and Thompson 1980) from leavesof 25-day-old green-house-grown Pusa Basmati 1 plantswas used for PCR amplification of OsLOX3 gene. ThePCR products were separated on 1% agarose gel, and thesingle specific band of PCR was cloned into the pGEMvector (Promega) for sequencing.

Apart from induction of disease, plant response to stressand possible activation of OsLOXs was also examined uponphysical wounding. The third leaf of 25-day-old green-house-grown rice plants was wounded with a scalpel andsprayed with 100 μM of MeJ in 0.1% (v/v) ethanol along

266 Funct Integr Genomics (2012) 12:265–275

with water control applied externally (Kolomites et al.2000). Leaf samples were collected at 0 and 2 h after injuryfor semi-quantitative RT-PCR and RNA analysis.

Identification and isolation of rice OsLOX genes

For prediction of putative OsLOX genes in O. sativa indica(cv. Pusa Basmati 1) and japonica (cv. Nipponbare), LOXnucleotide sequences from Arabidopsis thaliana (TAIR acces-sions: AT1G17420, AT1G55020, AT1G67560, AT1G72520,AT3G22400, and AT3G45140) were downloaded and used tosearch against rice genome employing BLASTN, TBLASTX,andMEGABLAST (Altschul et al. 1990, 1997). Gene findingsoftware tools GENESCAN (Stormo 2000) and FGENESH(Solovyev et al. 2006) were employed to validate the structureof predicted OsLOX sequences. To investigate the role ofindividualOsLOX genes in plant defense, LOX3 gene sequen-ces inA. thaliana, pea, potato,O. sativa (japonica), and barleywere downloaded from GenBank. Multiple sequence analysisof these LOX sequences along with the isolated OsLOX3sequences in the current study employing ClustalW yieldedconserved sequence patterns. Primer pairs (Table 1 in theElectronic supplementary material (ESM)) were designed us-ing SeqBuilder tool from DNASTAR (http://www.dnastar.com/t-products-lasergene.aspx) for PCR amplification ofOsLOX3 gene and semi-quantitative RT-PCR amplificationof OsLOX cDNA sequences.

Densitometry analysis of gels for semi-quantitative analysisof expressed (LOX and tubulin) genes was done employingGene Profiler software (Alpha Innotech Corporation USA).Individual gels were scored and relative densitometry valuesfrom three independent gels were recorded. OsLOX cDNAexpression analysis was done using GraphPad Prism (version5.0). Secondary structures of isolated LOX3 protein andOsLOX cDNAs were modeled based on soybean LOX struc-ture template (PDB. 1Yge) employing PyMOL, v.1.2 (http://www.pymol.org/).

RNA isolation and semi-quantitative RT-PCR

Total RNAwas isolated from plant samples of Pusa Basmati1 and five independent RNA extractions were made from

five distinct stages of plant development (viz., root andshoot at 25 days, shoot at 60 days, and flag leaf at milkyand mature seed stages) and also from leaves after woundingand MeJ ectopic application and control plants, using RNeasymini kit (QiaGen), following manufacturer’s instructions.DNAsel-treated RNA samples were then reverse transcribedwith oligo(dT) primers for 60 min at 42°C. For semi-quantitative RT-PCR analysis, tubulin was used as a positiveinternal control. For the RT-PCR, 12.5 pmol of gene-specificprimer was used in a 25-μl reaction volume containing0.2 mM of each of dNTPs, 2 mM MgCl2, and 1 U GoTaqFlexi DNA polymerase (Promega) which is provided with agreen buffer containing gel loading dye so that the PCRproducts can be loaded directly onto the gels. The temperatureprofiles used for the PCR were 94°C for 5 min initial dena-turation followed by 35 cycles of 95 for 1 min, 58°C for 1 min,72°C for 1 min, and final extension for 10 min.

PCR amplification and cloning

Predicted putative OsLOX sequences were PCR amplifiedand sequenced. The amplified products were separated on0.7% agarose gel and the expected size amplicons were geleluted using QIAGEN-QIAquick gel extraction kit (QiaGen,USA) and cloned in pGEM-Teasy vector (Promega, USA) asper kit instructions. Eluted rice PCR amplicons of OsLOXgenes and cDNAs were directly sequenced. Of the threeisolated genes (OsLOX1, OsLOX2, and OsLOX3), OsLOX3was chosen in the present study to investigate its role duringblast pathogen attack.

Sequence analysis of OsLOX cDNA clones

Isolated and sequenced OsLOXs were BLAST searched tolocate homologous sequences in the databases. Domaincomposition of the OsLOX amino acid sequences was ana-lyzed to deduce functional information employing INTER-PROSCAN (v. 4.4, Quevillon et al. 2005). LOX proteinsequences from different crop species were downloaded frompublic repositories, compared, and aligned with OsLOXsequences using ClustalW (Thompson et al. 1994). To map

Table 1 Position of different OsLOX loci in rice genome

OsLOX/GenBank Ac. number Locus position OsLOX/GenBank Ac. number Locus position

OsLOX1 (FJ660618) AP008208 (5279133–5282623) OsLOX7 (FJ660630) AP008211 (13601100–13607113)






Funct Integr Genomics (2012) 12:265–275 267

http://www.dnastar.com/t-products-lasergene.aspx

http://www.dnastar.com/t-products-lasergene.aspx

http://www.pymol.org/

http://www.pymol.org/

the location of identified OsLOX genes, GenomeBlast searchwas carried out using individual OsLOX genes as querysequences. Upstream (1,000 nt) and downstream (200 nt)regions of isolated OsLOX cDNAs were mined to locateconserved motifs located in the promoter regions employingPLACE (Higo et al. 1999) and PlantCARE (Lescot et al.2002). To locate co-occurring motifs that are over-represented in the promoter regions, full-length genes from

japonica (EU146294), indica (EU700314), A. thaliana(AEE29585.1), and potato (Y18548.1) were compared. Weconsidered those motifs occurring in at least 50% of OsLOXpromoters with a p value of ≤0.03 as over-represented motifs.Thus, gatheredmotifs of variable length were searched againstRice Transcription Factor Database (http://drtf.cbi.pku.edu.cn) to understand the expression patterns of individualOsLOX cDNAs and their physiological roles. OsLOX genes

Mar

ker-

1kb

Lip

oxy

gen

ase-

1

Lip

oxy

gen

ase-

2

Lip

oxy

gen

ase-

3

3866bp

6112bp

3532bp

(A) (B)

Fig. 1 a PCR amplification of OsLOX1, OsLOX2, and OsLOX3 genes in O. sativa (cv. Pusa Basmati 1). b Predicted structure of OsLOX3(EU700314), Pusa Basmati 1 (FGENESH, v.2)

A.thaliana O. sativa (cv. Pusa Basmati 1)

Fig. 2 Synteny relations of OsLOXs between indica rice and A. thaliana


http://drtf.cbi.pku.edu.cn

http://drtf.cbi.pku.edu.cn

and the underlying synteny relationships were deduced bycomparing chromosomes of indica rice and Arabidopsisemploying Artemis and ACT tools (Carver et al. 2008).

Results

Identification and isolation of rice LOX genes and OsLOXcDNAs

Sequences of six LOX genes in A. thaliana (accessionsAT1G17420, AT1G55020, AT1G67560, AT1G72520,AT3G22400, and AT3G45140) were downloaded fromTAIR. Sequence homology search with these Arabidopsissequences against rice genome database employing MEGA-BLAST (Altschul et al. 1990, 1997) enabled to locateOsLOX orthologs in japonica and indica rice. TwelveOsLOX genes were located in both japonica (cv. Nippon-bare) and in indica (cv. Pusa Basmati 1) of O. sativa dis-tributed across different chromosomes. These sequenceswere deposited in GenBank (Table 1; Table 2 in the ESM).Genomic presence of predicted OsLOX cDNAs was con-firmed by semi-quantitative PCR amplification and tissue-specific expression analysis.

Gene-finding software tools GENESCAN (Stormo 2000)and FGENESH (Solovyev et al. 2006) were employed tovalidate the presence of predicted coding sequences (Fig. 1in the ESM). The genomic structure of the full-lengthOsLOX3 sequence was deduced comparing the cDNA andgenomic sequences. FGENESH prediction enabled to pre-dict the structure of OsLOX3 full-length sequence in indicarice (cv. Pusa Basmati 1, GenBank accession EU146294)spanning 3,532 bp with three exons (Fig. 1b).

Comparison of chromosomes between A. thaliana andrice for OsLOX genes has revealed interesting synteny rela-tionships. Genes of chromosome 3 from Arabidopsisshowed co-linearity with 2, 4, 5, 11, and 12 of rice chromo-somes; whereas, Arabidopsis chromosome 1 revealed co-linearity with rice chromosomes 3 and 8 (Fig. 2). IsolatedOsLOX3 gene was located on the long arm of chromosome11.

Sequence analysis

Isolated indica rice OsLOX sequences were comparedamong one another and to OsLOXs in japonica and A.thaliana. Pairwise sequence analysis of OsLOX proteins inindica with japonica rice revealed very high levels of aminoacid conservation up to 100%. Only OsLOX8, OsLOX9,and OsLOX12 exhibited variation in amino acid composi-tion with homology ranging from 67.5% to 91.9% (Table 2).Similar patterns of amino acid conservation was observedbetween indica rice and A. thaliana (data not shown)T

able

2OsLOX

proteinsequ

ence

similaritiesbetweenO.sativajapo

nica

andO.sativaindica

(MATGATou

tput)

O.sativaindica

vs.O.sativajapo

nica

OsLOX1

OsLOX2

OsLOX3

OsLOX4

OsLOX5

OsLOX6

OsLOX7

OsLOX8

OsLOX9

OsLOX10

OsLOX11

OsLOX2

OsLOX1

94.4

32.5

32.4

33.3

31.5

33.3

30.6

25.3

31.1

26.8

5829

.6

OsLOX2

30.3

92.3

36.7

34.1

4535

.835

.831

.846

.830

.928

.236

.7

OsLOX3

35.2

3510

031

78.4

60.8

3928

.629

.532

.930

.531

.4

OsLOX4

31.7

33.3

33.3

100

33.3

31.7

3133

.129

.529

.537

.433

.3

OsLOX5

30.6

41.9

78.4

33.3

100

56.9

42.9

30.5

34.5

30.9

28.2

31.3

OsLOX6

32.4

3560

.831

.756

.910

030

.531

.232

.428

.932

.132

.4

OsLOX7

31.5

33.3

3931

42.9

30.5

100

31.2

32.4

35.6

29.8

33.3

OsLOX8

28.6

35.9

38.4

42.1

36.6

34.8

35.1

67.5

26.6

26.2

29.8

29.5

OsLOX9

28.1

51.1

30.2

33.8

33.8

30.9

30.9

32.5

88.5

29.5

37.4

30.2

OsLOX10

28.9

33.6

32.9

28.9

30.9

28.2

35.6

27.3

28.2

100

35.6

28.2

OsLOX11

5832

.130

.537

.427

.532

.129

.831

.240

.335

.610

028

.2

OsLOX12

25.9

33.3

32.4

33.3

34.3

34.3

32.4

33.8

28.1

30.2

2991

.9


ranging from 56.8 (OsLOX3) to 64.8 (OsLOX6). However,not very high level of similarity was observed among allisolated indica OsLOXs (cv. Pusa Basmati 1) which mostlyranged from 50% to 60% from pair-wise alignment (Table 3in the ESM). Highest level of similarity was observed forOsLOX5 with OsLOX3 (82.4%) and OsLOX (81.0%).

Domain composition was analyzed in all 12 OsLOXprotein sequences and OsLOX3 protein. INTERPROSCANresults revealed presence of two domains-LH2 (LOX, ori-ented towards C terminal) and polycystin-1, LOX, and alphatoxin (PLAT; towards N terminal) in all the analyzedsequences (Fig. 3). PLAT domain spans from 119 to 220amino acids of OsLOX3 in A. thaliana and 58 to 161 aminoacids in Pusa Basmati 1; whereas, LH2 domain starts from100 to 755 residues in A. thaliana and stretches from 232 to903 residues across OsLOX3 sequence in Pusa Basmati 1.

All the isolated rice OsLOXs were found to contain LH2and PLAT domains that are essential for catalysis and oxy-gen binding. Protein domain analysis and multiple sequencealignment has revealed that all isolated OsLOXs showedfunctionality with LOX super family. In all the OsLOXs,LOX domain chiefly harbored high proportion of hydropho-bic amino acids (85% to 91%) and are oriented towards C

terminal. In O. sativa (cv. Nipponbare), LOX domain inOsLOX1and OsLOX3 sequences were found to consist highlevel of hydrophobic residues (80% and 70.9%, respectively).In OsLOX3, an interesting residue pattern of five histidineswas observed. High levels of hydrophobic residue composi-tion was also observed in Arabidopsis and O. sativa indicafrom ClustalWalignment. It is interesting to note that in all theanalyzed OsLOXs motifs, His X4, His X8, and His X17 areconserved in the hydrophobic residues of the LOX domain(Fig. 3).

Modeled secondary and three dimensional structures ofisolated OsLOX3 (Fig. 4a) and of all other OsLOXs (Fig. 2in the ESM) were compared with soybean LOX1 (PDB.1Yge) template using PyMol and Swiss PDB viewer tools.Molecular weight of LOX3 protein was found to be88.5 kD, and topology analysis revealed it as a membranebound protein with four helices spanning the surface. Pro-tein structural similarities observed in isolated OsLOXswere compared with soybean template structure (PDB.1Yge) and they ranged between 55% (OsLOX2) and 71%(OsLOX9). Similarly, a significant variation of secondarystructural elements among members of all isolated OsLOXswas observed compared with soybean template structure

Fig 3 Multiple sequence alignment of 12 OsLOX cDNAs with the conserved motifs in LOX domain (displayed inside red boxes)

Fig 4 a Secondary structure ofLOX3 protein of Pusa Basmati1 (PyMOL, v.1.2). Helices(cyan), sheets (magenta), andcoils (salmon). b LOX3 proteinmodel from O. sativa indica(distance between Pro33 andIleu74→8.7 Å and Va21→20 Å, marked in red )


(PDB. 1F8N). The significant differences in secondarystructural elements observed include: H-bonds (297 to528), helices (23 to 42), β sheets (11 to 42), and turns (51to 105) in OsLOX cDNAs. Among the isolated riceOsLOXs, significantly lower number of H-bonds (297),helices (23), β sheets (11), and turns (51) were observedin OsLOX12. Visible differences were also observed (Fig. 2in the ESM) in arrangement of barrels (β sheets painted inyellow). The active site region of the isolated rice OsLOXproteins (OsLOX1, OsLOX2, OsLOX3, OsLOX6, andOsLOX11) occupied significantly large spaces where atleast one hydrophobic amino acid stood apart from rest ofthe residues at a distance. In OsLOX3, for example, Valine(at position 21) stood apart from other amino acids at adistance of 20°Å (Fig. 4b).

Promoter regions of OsLOX cDNAs were analyzed tolocate various motifs and understand their regulatory rolesin gene expression in rice plant development. Computation-al analysis of upstream and downstream regions of genes,and identification of Cis-regulatory elements was performedby using databases PLACE and PlantCARE. Upstream(1,000 nt) and downstream (200 nt) regulatory regions weremined and conserved motifs located in the promoter regionsviz. (−1,000 and −800), (−800 and −600), (−600 and −400),(−400 and −200), (−200 and −1), and (+1 and +200) werescored. The highest percentage of the motifs was found inthe region stretching from +1 to +200. The results ofdetected motifs are shown in Table 3. Interestingly, knownand unknown motifs of variable lengths were located in thepromoter sequences of LOX genes. Motif detection toolMEME (Bailey and Elkan 1994) was employed and motifswith a width between four and ten nucleotides with anynumber of repetitions were mined. The motif search wasrestricted to the regions of the given strand and was limitedto 70. A total of 50 motifs were detected in the promoterregions of 12 LOX genes. The significance of these motifswas evaluated employing PlantCARE and PLACE. Interest-ingly, some motifs seemed to prefer specific genomicregions in abundance. We compared promoter regions of

OsLOX3 genes in japonica and indica rice and Arabidopsisfor possible location of orthologous motifs that are overrepresented. Abundance of motifs CGTCA and TGACG(p≤0.03) in the promoter region of isolated OsLOX3 gene(EU146294) in rice is interesting (Table 3). Similarly, the200-nt upstream region of OsLOX3 gene orthologs in Ara-bidopsis, O. sativa japonica and O. sativa indica also con-firmed the presence of motifs CGTCA and TGACG inabundance. Over-represented motifs present in variousOsLOX cDNAs that were shown to have expressed inspecific rice developmental stages in tissues (Fig. 3) weresearched against TRANSFAC database. Individual motifsspecific to tissue expression were selected. TRANSFACDatabase search enabled classification of over representedmotifs in OsLOX cDNAs in to different functional catego-ries ranging from biotic stress mitigation (in majority ofcases response to MeJ signaling), embryo development totranscriptional regulation. Thus the generated informationpartially aided in analyzing the observed variations in ex-pression of rice OsLOX cDNAs and defining various phys-iological roles played by them.

Gene expression analysis

Expression analysis of 12 isolated OsLOX cDNAs wascarried out from different tissues of rice plant viz., rootsand shoots of 25-day-old seedlings, 60-day-old plants, flagleaves, early grain development (milky stage), and matureseeds. Substantial levels of OsLOX3 RNA were detected(Fig. 5) in both 25- and 60-day-old rice plants (i.e., seedlingand active tillering stages). In 25-day-old plants, RNA fromOsLOX2, OsLOX5, OsLOX8, OsLOX9, and OsLOX 11were observed in shoot at a substantial level and was unde-tected in the rest of the OsLOXs. In 60-day-old plants(active tillering stage), abundant RNA levels were observedfrom all isolated OsLOXs except OsLOX4 and OsLOX10.At milky stage of grain development, RNA was observedfrom all the OsLOXs. Only OsLOX1, OsLOX2, OsLOX5,OsLOX11, and OsLOX12 were found to be expressed in

Table 3 Cis-acting elements predicted in regulatory regions of isolated OsLOXs


Fig 5 Expression profiling of LOX genes of different stages of development (root, 25 days old; shoot, 25 and 60 days old; and flag leaf anddeveloping seed, milky stage)


flag leaf. In case of OsLOX3, RNA levels were observed inall the developing stages except flag leaf.

MeJ perceives and responds to local and systematic sig-nals generated by external stimuli such as wounding andattack of pests and pathogens (Cheong and Choi 2003;Creelman et al. 1992). Hence, effects of pathogen-causedwounding and of physical wounding were examined onpossible induction of OSLOXs. Plant response to woundingwas examined by physical wounding and subsequent appli-cation of ectopic MeJ on wounded leaves of rice plantsgrown in green house. Rice leaves were wounded with ascalpel and sprayed with 100 μM of MeJ or water control,and their transcription profiles were determined after “0”and “2”h of MeJ application. RT-PCR transcription profilesof the wounded leaf samples (Fig. 6) revealed differentialpatterns of expression. Samples from OsLOX1, OsLOX2,OsLOX5, OsLOX11, and OsLOX12 showed no change inexpression levels between 0 h (or basal) and after 2 h ofwounding similar to control un-injured plants. Samples fromOsLOX3, OsLOX6, OsLOX8, and OsLOX9 showed signif-icantly increased transcription levels 2 h after wounding,samples from OsLOX4 and OsLOX7 showed traces of tran-scripts and in case of OsLOX10 transcripts were notdetected even 2 h after wounding and MeJ application.

Discussion

OsLOX3 gene and OsLOX cDNAs isolated in present studyshared major features characteristic to LOX super family.Sequence analysis of isolated gene coded OsLOX proteins

demonstrated significant levels of similarity and variation atboth sequence and structure levels. OsLOX3 contained cat-alytic LOX domains much required for oxygen binding andsynthesis of oxylipins. Sequence comparison of OsLOX3(Pusa Basmati 1) with japonica rice showed very high levelsof conserved amino acid sequences ranging up to 100%except for OsLOX8, OsLOX9, and OsLOX12 that rangedfrom 67.5% to 91.9%. Pair-wise sequence analysis of LOX3showed very high levels of amino acid similarity also withother plant LOXs (StLOX, 53%; LeLOX3, 49%; GmLOX3,49%; AtLOX3, 56.8%; and ZmLOX3, 52%). Similar con-served patterns of LOX aminoacid was reported in A. thali-ana by Bell and Mullet (1993) and Melan et al. (1993).

Isolated rice OsLOX3 and OsLOX cDNA proteins weremodeled and used to compare the variations at sequence andstructure levels. Modeled indica rice OsLOX3 primarilycontained two domains: LH2 catalytic domain located onC terminal side (consisting 20 helices) and PLAT domaintowards N-terminal (consisting chiefly of β sheets). But inOsLOX3, the PLAT domain was observed to be sandwichedbetween two antiparallel β sheets and the sheets appear tobe smaller than that of the soybean template. Unlike insoybean, significantly large spaces were observed in theactive site region of OsLOX3, where at least one hydropho-bic amino acid is observed to be placed at a significantdistance from the remaining residues. In OsLOX3 it wasobserved that valine (at position 21) stood apart from otheramino acids at a distance of 20 Å. Interestingly in thepresent study modeled structure of OsLOX1 was similar toOsLOX3 and also showed response upon wounding similarto that caused by attack by pests and pathogens. It is wellknown that mode of arrangement of β barrels (sheets) playsan important role in active site affinity (Shen et al. 2008).Visible variations in arrangement and length of sheets wereobserved among different OsLOX cDNA structures in ourstudy. Presence of a cluster containing six serially arrangediron-binding histidine residues in the PLAT and LH2domains was observed in OsLOX3. Histidines are essentialfor catalysis and addition of oxygen to free fatty acids. Inhuman LOXs-enhanced enzyme, activity of group LOXswas reported due to presence of a similar pattern of histi-dines that play important role in iron binding in oxygenationof fatty acids (Zhang et al. 1992). PLAT domain found in avariety of membrane or lipid-associated proteins in eukar-yotes (Minor et al. 1996) mediates membrane attachmentvia other protein binding partners which is possibly requiredin transduction of perceived external signals to nucleus fortranscription of pathogen-related “PR” genes in attack bypathogens apart from its major role in lipid oxidation. Thusthe elucidated protein structure information establishes therole of LOX proteins in imparting disease resistance in rice.The role of LOX proteins and also the LOX genes (asdescribed below in expression and promoter analysis) in

Fig 6 Expression profile of wounded leaves of OsLOX cDNAs. RT-PCR amplification profiles after 0 and 2 h of ectopic application ofMeJ


wound response after attack of pathogens is also confirmedupon ectopic application of MeJ in the present study. How-ever, we presume further investigations are needed to asso-ciate observed similarities at the level of sequence alignmentand structure with the presumed physiological role playedby individual OsLOXs in rice plant development.

Chromosomal syntenic relationships between Arabidop-sis and rice OsLOX3 (together with OsLOX11 andOsLOX12) showed significant similarity with AtLOX2.The predicted sequences were mapped on the chromosomesof japonica and indica rices and compared with positions inA. thaliana. OsLOX3 was observed to be located on thelong arm of chromosome 11, in company of other knowndisease resistant genes. Location of OsLOX3 gene, demon-strated to be a major player in imparting resistance againstM. grisea in the present study assumes interest for ricebreeders who often prefer transfer of closely located genesfor development of disease-resistant varieties.

Gene expression analysis revealed tissue specific expres-sion of OsLOX3 gene during rice plant development. Sub-stantial levels of RNA expression of OsLOX3 gene appearsto be confined to roots, shoots and the milky stage of thedeveloping seeds but not in flag leaf. Similarly activation ofOsLOX genes was earlier reported up on attack by patho-gens (Peng et al. 1994) and insects (Ohta et al. 1992; Wanget al. 2008) in japonica rice and in soybean (Creelman et al.1992). Rice plants inoculated with virulent strains of M.grisea and external application of MeJ on wounded leavesrevealed increased levels of RNA synthesis and successfulamplification of OsLOX3 gene.

To examine the influence of external stimuli during path-ogen attack, promoter region analysis of LOX3 gene andother OsLOXs was done. In the upstream region of OsLOX3gene and other OsLOXs, motifs CGTCA and TGACG wereobserved to be over-represented. Both these motifs areknown to be associated with regulation of associated syn-thesis of MeJ during plant disease and stress responses(Després et al. 2003; Kesarwani et al. 2007). Bu et al.(2008) reported role of transcriptional factor AtMYC2 mu-tant (A. thaliana) in induction of LOX2 and PR genesinvolved in response to wounding (both in mechanical andbiotic). Activation of LOX1 and LOX3 from potato werereported to be associated with wound response in attack bypathogens (Kolomites et al. 2000) and after wounding insoybean (Saravitz and Siedeow 1995). Abundance ofCGTCA, ABRE, SKN-1, ARE Cis-regulatory elements inpromoter regions of isolated OsLOX cDNAs led to associ-ate them with potential OsLOX functions such as rice grainflavor and abiotic stress during rice plant development.

To our knowledge, this is the first report on isolation of afull-length OsLOX3 gene in indica rice and its demonstratedrole in blast pathogen infection and upon physical wound-ing. The study may contribute to the ongoing efforts by rice

breeders in development of blast disease resistant rice varieties.However, further detailed investigations are needed to explainthe varied roles played by OsLOXs in rice plant.

Acknowledgments The authors are grateful to Bioinformatics Infor-mation Network under Department of Biotechnology, Government ofIndia for grant provided to support experimental costs and fellowshipto VKS. Also, the authors are thankful to B. Murugan and A. Dev forhelp in disease inoculation experiments in the green house and toDepartment of Molecular Biology and Genetic engineering, G.B. PantUniversity of Agriculture and Technology, Pantngar, India for provid-ing all laboratory and green house facilities.

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LOX genes in blast fungus (Magnaporthe grisea) resistance in rice