Industrial Crops and Products 45 (2013) 2024

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Industrial Crops and Products

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Castor (Ricinus communis L.) Rc-LOX5 plays important role in wilt resistance
Somnath D. Mhaske a , Mahesh Kumar Mahatma a, , Sanjay Jha a , Pushpendra Singh b , Lalit Mahatma b , Vipul B. Parekh a , Taslim Ahmad a
a b

Department of Plant Molecular Biology and Biotechnology, Navsari Agricultural University, Navsari 39450, Gujarat, India Department of Plant Pathology, Navsari Agricultural University, Navsari 39450, Gujarat, India

a r t i c l e

i n f o

a b s t r a c t
Phyto-oxylipins are a group of biologically active molecules that play an important role in plant defense. Their production begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOXs; EC 1.13.11.12) to form 9- or 13-hydroperoxides. These are substrates for several enzymes involved in the synthesis of nal oxylipins, which can act as signal molecules and/or direct antimicrobials. Recent completion of the castor bean genome sequence now permits genome-wide analysis of the LOX gene family in castor as well as comparison with LOX in Arabidopsis. We identied 12 candidate LOX genes in the castor bean genome. Phylogenetic analysis indicated that these LOX members cluster into two groups, designated types 1 and 2, as expected from previous studies. Out of which six LOX gene specic primers were designed to amplify castor LOX genes i.e. LOX1, LOX2, LOX3, LOX4, LOX5 and Dox. Sequence analysis showed conserved ve iron binding sites in the LOX domain of all the Rc-LOX, however, only LOX5 contained consensus (positions 547, 556, and 715) histidines residue. Expression analysis of LOX2, 3, 4, 5 and DOX genes in resistant and susceptible genotypes of castor at 0 days after infection (DAI), 5 DAI and 10 DAI (30 days after sowing) was carried out using quantitative real time (RT)-PCR during castor beanFusarium oxysporium f. sp. ricini interaction. Results suggest that 2 (LOX2 and LOX5) of 6 Rc-LOX genes were detectable. Resistant genotypes (48-1 and SKP-84) exhibited appreciably higher expression of LOX5 during castor beanF. oxysporium interaction, which further suggest the participation of Rc-LOX5, a type-1 LOX predicted to be 9-LOX, in wilt resistance. 2012 Elsevier B.V. All rights reserved.

Article history: Received 18 September 2012 Received in revised form 22 November 2012 Accepted 23 November 2012 Keywords: Castor Expression analysis Gene Lipoxygenase Wilt Resistant genotype

1. Introduction Castor (Ricinus communis L.) is highly remunerative industrially important non-edible oilseed crop belongs to family Ephorbiaceae. Castor seeds contain about 4655% oil by weight. It is a raw material for paints, coatings, inks, lubricants and a wide variety of other products. Castor cake not suitable as an animal feed because of the presence of toxic protein called ricin which is commonly referred to as CBA (castor bean allergen), however, is widely used as organic manure in agriculture. None of the toxic components are carried into the oil, therefore, castor oil is used as laxative in human medicine. Another product formed from the modication of castor oil is sulfated castor oil which an active wetting agent and used extensively in dyeing and in nishing of cotton and linen (Ogunniyi, 2006). The major castor growing countries are India, China, Brazil and Russia. In India, Gujarat is leading castor growing state, which

Corresponding author. Present address: Directorate of Groundnut Research, Indian Council of Agricultural Research, Post Box No. 5, Junagadh 362001, Gujarat, India. Tel.: +91 285 2673041x135; fax: +91 285 2672550. E-mail addresses: maheshmahatma@nrcg.res.in, maheshmahatma@gmail.com (M.K. Mahatma). 0926-6690/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.11.035

produces 82% of total production in India. Wilt disease of castor bean caused by Fusarium oxysporum f. sp. ricini is a major constrain of crop production in India. Monocropping has been followed due to its high economical return, which has established an endemic situation in the major castor growing area of Gujarat (Dange et al., 1997). More than 80% loss in crop yield have been reported due to the diseases compelling farmers to grow another crops which are less remunerative (Singh et al., 2011). Resistance source is available in the local germplasm and is being introgressed in the elite germplasm. The disease is often observed in the compatible interaction of host and pathogen. However, resistance germplasm contains some gene responsible for the incompatible interaction. Lipoxygenase (LOX) gene is an important gene of incompatible interaction. The role of LOX in plantmicrobe interactions was rst time realized when arachidonic acid and eicosapentaenoic acid were isolated from the mycelium of a phytopathogenic fungi Phytophthora infestans, which was shown to elicit phytoalexin accumulation and hypersensitive cell death in potato tissues, probably via LOX action. Transgenic tobacco plants expressing antisense LOX clearly demonstrates the role of LOX in plant resistance to pathogens (Rance et al., 1998). The enzyme is able to generate molecules that can be divided into three groups with different functions: (a) hydroperoxides and free radicals that

S.D. Mhaske et al. / Industrial Crops and Products 45 (2013) 2024 Table 1 Sequence of primers used for sequencing and expression analysis. Primer LOX1 F LOX1 R LOX2 F LOX2 R LOX3 F LOX3 R LOX5 F LOX5 R Dox F Dox R LOX4 F LOX4 R RcUBIQ F RcUBIQ R Sequence 5 -GGATTACTACATTCACTCCTGCAAC-3 5 -GGCAGATATGCCTTGTTAGTAAAGA-3 5 -CAAGATGCAACACAGATACACTTTC-3 5 -TCTTTCCAACCTTGTTCTTTAAGTG-3 5 -GGAATTAGAAAATTGTGCGATAGAA-3 5 -CAGAGACTTCTATTCTTCTGCCATC-3 5 -AAGTATTACTCCAATAACCGCTGTG-3 5 -CATGGAAGGTAGGTCTTGTTAGAGA-3 5 -GATCTAACGGATGACAAAGAAGCTA-3 5 -ACTTTCTGTGGTATTCACCCATTTA-3 5 -ACAGATGACACTAAGGAGTCTCCAG-3 5 -TGGAACATTCTTTCTTTGACTTTTC-3 5 -TCT TCT TAG GCC TTA ACT GAT TGC-3 5 -ATG GCT ATG GCT GGA TTG TACC-3 NCBI No. XM 002512340.1 XM 002513397.1 XM 002519024.1 XM 002516725.1 XM 002517356.1 XM 002519026.1 XM 002530294.1

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might be involved in the localized cell death observed during the hypersensitive response; (b) signal molecules such as jasmonic acid (JA) and its methyl ester that can trigger defense gene expression and amplify the initial response; and (c) antimicrobial compounds such as 2-trans-hexenal that constitute a direct defense against pathogen attack (Croft et al., 1993). Diverse roles played by different LOXs during growth, development, and environmental stress can be explained by the versatile catalytic activities of LOXs and region specicity of various oxylipins in plant tissues (Gao et al., 2011). Recently, 12 putative OsLOX genes were studied in Oryza sativa indica. Gene expression, amino acid composition, protein secondary structure, and promoter region analysis revealed the role of OsLOX3 gene in providing resistance to blast fungus (Magnaporthe grisea) in rice plants (Marla and Singh, 2012). Nevertheless, there is no information on LOX gene analysis and expression during castorFusarium oxysporium f. sp. ricini interaction. Therefore, these observations reinforced to sequence LOX genes with further characterization at transcriptional level to conrm which LOX gene is important for castor wilt resistance. 2. Materials and methods 2.1. Castor genotypes and its inoculation with F. oxysporium f. sp. ricini The investigation was carried out using the four castor genotypes. The seeds of two resistance genotypes (SKP-84, 48-1) and two susceptible genotypes (VP-1, VI-9) to castor wilt were procured from the Main Castor and Mustard Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardar Krushinagar, Dantiwada, Gujarat, India. Seeds were sown on the surface of the ne sterilized sand and then covered with a 2 cm layer of sand. The crop was well irrigated regularly to avoid any physiological stress and to maintain high relative humidity condition. Highly susceptible genotypes VP-1, VI9 and resistant genotypes SKP-84, 48-1 were sown in individual trays. All the four genotypes were infected with F. oxysporium f. sp. ricini by mechanical method at 20 days after germination. Plants were pulled out gently from the sand and roots were washed with distilled water then roots from terminal sites about 12 cm were slotted out. Injured roots of castor seedlings were dipped in 1 106 fungal suspension for 1015 min, while for control treatment the injured roots were dipped in distilled water. After that plants were transplanted in fresh sterilized pot containing

sterilized soil and sand in 1:1 ratio. For the relative expression analysis of lipoxygenase genes, second fresh leaves were taken at 0, 5 and 10 days after infection (DAI) from both infected and noninfected plants, while for the sequencing of LOX genes leaves of resistant genotype 48-1 was used after 20 days of germination. 2.2. RNA isolation and cDNA synthesis Total RNA was extracted from infected and non-infected leaves with TRIzol reagent. RNA concentration was measured spectrophotometrically at 260 nm. The rst strand cDNA synthesis reactions were performed using the Omniscript RT Kit (Qiagen) with oligodT 18 primers according to the manufacturers instructions. 2.3. Amplication of lipoxygenase gene(s) Total 12 LOX genes in castor were reported by Thakur et al. (2011) out of these, 6 gene sequences were used to designed gene specic primers. Primers were designed using primer 3 software (Table 1) and used to amplify each cDNA by RT-PCR. The PCR amplication of lipoxygenase genes were performed using different reverse and forward primers i.e. LOX1, LOX2, LOX3, LOX5, DOX, LOX Putative (Table 1). The reaction mixtures composed of 5 l 10 PCR buffer, 0.2 mM dNTPs, 1 M each primer, 200 ng cDNA template, 2.5 units Taq DNA polymerase and sterile deionized water to a nal volume of 50 l. PCR programme was as follows: initial denaturation at 94 C for 5 min, followed by secondary denaturation (94 C, 30 s), annealing (65 C, 45 s), initial extension (72 C, 1 min), and nal extension at 72 C for 5 min with 31 cycles. PCR product was stored at 4 C for future use. All the PCR amplied products were run on 0.7% agarose gel containing 5 l ethidium bromide, in 0.5 TBE (5 TBE stock, 54 g Tris, 27.5 g boric acid, 20 ml of 0.5 M EDTA, pH8.0). Amplied product (5 l) was mixed with 1 l of 5 gel loading dye and loaded in the well. The electrophoresis was carried out at 60 mA (constant) to separate the amplied bands. The standard DNA marker (DNA ladder, 100 bp) was also run along with the samples. The single band were seen under UV light and photographed by gel documentation system. 2.4. DNA sequencing Cycle sequencing of the PCR product was done using Big Dye sequencing kit 3.1v (Applied Biosystems). A 250 ng of the PCR product was used for cycle sequencing. Product of cycle sequencing (10 l) was puried by mixing 12 l of master mix I consisting 10 l

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Fig. 1. Amplied product of cDNA with (a) LOX5 and (b) Ubiquitin primer 1, 3, 5 and 7 non-infected leaf cDNA of VP-1, VI-9, 48-1 and SKP-84 at 5 DAI 2, 4, 6 and 8 infected leaf cDNA of VP-1, VI-9, 48-1 and SKP-84 at 5 DAI 9, 11, 13 and 15 non-infected leaf cDNA of VP-1, VI-9, 48-1 and SKP-84 at 10 DAI 10, 12, 14 and 16 infected leaf cDNA of VP-1, VI-9, 48-1 and SKP-84 at 10 DAI M, molecular eight marker (Fermentas # SM0313); DAI, days after infection.

Milli-Q H2 O and 2 l of 125 mM EDTA and 52 l of master mix II consisting 2 l of 3 M NaOAc (pH 4.6) and 50 l of ethanol. Reaction mixture was incubated at RT for 15 min. and centrifuged at 12,000 g for 20 min at RT. Supernatant was decanted and washed with 250 l of 70% ethanol by centrifugation at RT for 10 min. Pellet was dried and dissolved in 15 l of Hi-Di Formamide for denaturation of DNA. Samples were snap chilled and then 10 l of sample was transferred to 96-well sequencing plate. Sequencing was performed using AB prism 3130 Genetic Analyzer (Applied Biosystems) with 4 capillaries.

2.6. Sequence analysis Sequences obtained by different gene specic primers were BLAST (Basic Local Alignment Search Tool) online on NCBI (National Center for Biotechnology Information) to nd the similarity between sequences available on NCBI database. Multiple sequences of nucleotides and proteins were aligned by CLUSTALW and phylogenic tree of sequences were constructed using PHYLIP 3.69. Dnadist (for DNA) or Protdist (for protein). WASP 2.0 software (ICAR, GOA) was used for analysis of gene expression data. Statistical analysis was performed using two factorial complete random design. Data were analyzed by ANOVA and means compared by least signicant difference test at 0.05 level of condence.

2.5. Expression analysis of lipoxygenase gene(s) Polymerase chain reactions were carried out in 96-well in a 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix with various LOX genes (LOX-2, LOX-3, LOX-5, Dox and LOX Put). As a control, the primer specic to castor Ubiquitin gene was used to normalize each sample for variations in the amounts of RNA used. RT-PCR was carried out in a 25 l reaction mixture containing 12.5 l 2 QuantiFast SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA), 10 pmol each reverse and forward primers (1 l each), rst strand cDNA 5 l (100 ng/reaction) and RNase free water 5.5 l. The reaction cycling programme consisted an initial denaturation for 5 min at 95 C, 35 cycles at 95 C for 10 s, and annealing at 60 C for 30 s. Each sample was tested in triplicate for all primers. Melting curve analysis was performed on all samples to ensure amplication of a single product with the expected melting temperature and the absence of primerdimers. A negative control without a cDNA template was run with each analysis to evaluate the overall specicity. The products of each primer set were tested by agarose gel electrophoresis to verify that a single product of the expected size was produced. Relative RNA quantities were determined with the deltadelta ( )Ct, according to the following formula (Dussault and Pouliot, 2006) comparing the data for each gene of interest with the data for mock-inoculated control samples at each time point. The data was normalized by comparison to reference gene. Ct = [(Ct G.O.I. Ctr Ct Ref. Ctr) (G.O.I. infected Ct Ref. infected)] where G.O.I. is the gene of interest, Ref. is the reference gene (Ubiquitin), Ctr is the control (non-infected) and fold increase = 2 Ct .

3. Results and discussion 3.1. Amplication of LOX genes and its analysis Total twelve LOX genes are available on the Castor DB (Thakur et al., 2011). Out of which six LOX gene specic primers were designed to amplify castor LOX genes i.e. LOX1, LOX2, LOX3, LOX4, LOX5 and Dox. All these designed primers could successfully amplify the cDNA of castor resistant and susceptible varieties for F. oxysporum f. sp. ricini (Fig. 1). Amplied products by all the primers of resistant genotype 48-1 were puried, sequenced and analyzed on BLAST search tool available at NCBI. Among all the genes LOX3, LOX5, and LOX4 showed more than 90% identity with castor LOX genes whereas Dox gene gave 97% identity with unknown gene of oxidoreductase class. Translation of the unique open reading frame present in LOX2, LOX3, LOX4 and LOX5 indicated that they codes for a protein of 900, 837, 868 and 786 amino acid long with an estimated molecular mass of 101.90, 95.03, 98.94 and 89.24 kD, respectively. Analysis of the deduced amino acid sequences of LOX2, LOX3, LOX4 showed conserved LOX domains for plant LOX-related proteins: the PLAT (for polycystein-1, LOX, -toxin) or LH2 (for lipoxygenase homology 2) domain. It consists of an eight stranded beta-barrel. The domain can be found in various domain architectures, in case of lipoxygenases, alpha toxin, lipases and polycystin, but also as a single domain or as repeats. The putative function of this domain is to facilitate access to sequestered membrane or micelle bound substrates. Interestingly, analysis of sequences of LOX5 identied PLAT/LH2 domain of plant lipoxygenase related proteins. The generally proposed function of PLAT/LH2 domains is to mediate

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Fig. 2. Conserved histidine (h) residue in the LOX5 (9-LOX) gene of castor at positions 547, 556, and 715.

interaction with lipids or membrane bound proteins. Lipoxygenases are nonheme, nonsulfur iron dioxygenases that act on lipid substrates containing one or more (Z,Z)-1,4-pentadiene moieties. In plants, the immediate products are involved in defense mechanisms against pathogens and may be precursors of metabolic regulators. Further analysis of LOX5 on KEGG (K15718) revealed that it is a 9-LOX (EC 1.13.11.58). LOXs are classied based on their positional specicity of active site of linoleic acid (LA) oxygenation. LA can be oxygenated either at carbon atom 9 (9-LOX) or at C-13 (13-LOX) of the hydrocarbon backbone forming the (9S)-hydroperoxy and the (13S)-hydroperoxy derivatives of PUFA (Feussner and Wasternack, 2002; Vellosillo et al., 2007). Moreover, amino acid composition revealed that only LOX5 protein (Fig. 2) contains conserved His (histidine) residues (at positions 547, 556, and 715) that are also observed in 9-LOX gene of cucumber and pepper (Hornung et al., 1999; Hwang and Hwang, 2010). LOXs and His have been implicated in iron binding and enzyme catalytic activity. The His residues that act as ligands to the active site iron are highly conserved in LOXs (Prigge et al., 1996). The other genes (LOX3 and LOX4) did not contain His residue at conserved position. Whereas, LOX2 contained His residue at 548, 553 and 717 position. Dendrogram of nucleotide (Fig. 3) and protein (Fig. 4) sequences of all LOX genes were constructed online using PHYLIP 3.69 programme on the basis of sequence alignment of genes, which showed LOX2 and LOX5 were found on separate cluster while LOX3 and LOX4 were on same cluster. These results might be due to absence of conserved His residue in both LOX3 and LOX4 and different position of His residue in LOX2 and LOX5. 3.2. Expression analysis of lipoxygenase gene(s) For further conrmation of the role of LOX genes in wilt resistance, expression analysis of lipoxygenase genes in response to F.

oxysporium f. sp. ricini interaction was examined in infected and non-infected leaves using 4 LOX gene specic primers i.e. LOX2, LOX3, LOX4 and LOX5 and one unknown gene of oxidoreductase. Expression of LOX2, 3, 4, 5 and DOX were induced 5 and 10 days after infection. Highest expression of LOX2 and LOX5 genes were observed (Fig. 5) in resistant genotypes at 5 DAI. Resistant genotypes also showed higher gene expression at 10 DAI. Both resistant genotypes (48-1 and SKP-84) exhibited appreciably higher expression of LOX5. The expression of LOX5 was maintained in 48-1 genotyped at 10 DAI. Expression of LOX2 was also higher in both resistant genotypes at 5 DAI however, the expression of LOX2 was signicantly lower at 10 DAI as compared to LOX5 in resistant genotypes. These results are suggesting that LOX5 (Rc9-LOX) is critically involved in the defense response. Similarly, 9-LOX activity and LOX1 mRNA expression are induced upon infection by Phytophtora parasitica var nicotianae in tobacco. Interestingly, both 9-LOX activity and LOX1 mRNA expression appear earlier in an incompatible plantpathogen interaction than in a compatible one, thus supporting a role for this 9-LOX in plant defense against fungal infection (Rance et al., 1998). Plant LOXs have been proposed to play a role in responses to wounding and JA, which triggers gene activation during wound response and necrotrophic fungal pathogen infection (Blee, 2002). Increased levels of RNA synthesis and successful amplication of OsLOX3 gene was observed in rice plants inoculated with virulent strains of M. grisea and external application of MeJ on wounded leaves (Marla and Singh, 2012). Infection with Arabidopsis brassicicola resulted in a resistance response in wild-type and CaLOX1-OX transgenic leaves. These resistance symptoms are consistent with the HR in Arabidopsis Col-0 plants infected by A. brassicicola (Narusaka et al., 2003). In contrast, LOX1 mutants were highly susceptible to A. brassicicola. The remarkable involvement of OsLOX3,

Fig. 3. Dendrogram of LOX genes on the basis of nucleotide sequences using PHYLIP 3.69 Dnadist programme.

Fig. 4. Dendrogram of LOX genes on the basis of protein sequences using PHYLIP 3.69 Protdist programme.

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Fig. 5. Expression of different LOX genes after infection with Fusarium oxysporum f. sp. ricini at 5 DAI and 10 DAI. CD 0.05 for gene genotype to compare expression at 5 DAI is 0.445 and at 10 DAI is 0.351.

CaLOX1 and AtLOX1 in mediating resistance to pathogen attack suggests that these LOX genes are highly conserved for disease resistance in plants. 4. Conclusions The results of present study show that LOX5 (Rc 9-LOX) gene consist all conserved regions of Histidine residues and maintain higher expression level in the resistant cultivar than susceptible cultivar to F. oxysporium f. sp. ricini. These results contribute the understanding of the role of LOX5 (9-LOX) in castor wilt resistance; other genes may be involved in other functions of LOX. The RcLOX5 gene can be used for screening or identication of wilt resistant genotypes. Acknowledgements Authors are thankful to the Research Scientist, Main castor mustard research station, Sardarkrushinagar Dantiwada Agricultural University, S.K. Nagar, Gujarat, India. References
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