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Planta (2014) 240:177–194DOI 10.1007/s00425-014-2073-7

OrIgInal artIcle

Elicitation of jasmonate‑mediated host defense in Brassica juncea (L.) attenuates population growth of mustard aphid Lipaphis erysimi (Kalt.)

Murali Krishna Koramutla · Amandeep Kaur · Manisha Negi · Perumal Venkatachalam · Ramcharan Bhattacharya

received: 5 March 2014 / accepted: 28 March 2014 / Published online: 26 april 2014 © Springer-Verlag Berlin Heidelberg 2014

genes. In contrast, when the jasmonate-mediated host defense was elicited by exogenous application of MeJ the treated B. juncea plants showed a strong antibiosis effect on the infesting aphids and reduced the growth of aphid populations. the level of redox enzymes cat, aPX, and SOD, involved in rOS homeostasis in defense signaling, and several defense enzymes viz. POD, PPO, and Pal, remained high in treated plants. We conclude that in B. jun-cea, the jasmonate activated endogenous-defense, which is not effectively activated in response to mustard aphids, has the potential to reduce population growth of mustard aphids.

Keywords Biotic stress · endogenous defense · Indian mustard · Methyl jasmonate · Mustard aphids

AbbreviationsnBS-lrr nucleotide-binding site–leucine-rich repeatrOS reactive oxygen speciesrt-Pcr reverse transcription polymerase chain

reactionSSH Suppression subtractive hybridization

Introduction

rapeseed-mustard (Brassica spp.)is considered to be the third most important oilseed crop in the world. In India, it contributes to 27.8 % of the national oilseed economy (Shekhawat et al. 2012). aphids, the hemipteran group of insects, are the major insect-pest of rapeseed-mustard in temperate and tropical agriculture. aphids damage the crop by diverting photosynthetic assimilates and vectoring numerous plant viruses (Hogenhout et al. 2008). though several defensive phytochemicals including indolic and

Abstract the productivity of Brassica oilseeds is severely affected by its major pest: aphids. Unavailability of resistance source within the crossable germplasms has stalled the breeding efforts to derive aphid resistant culti-vars. In this study, jasmonate-mediated host defense in Indian mustard Brassica juncea (l.) czern. was evaluated and compared with regard to its elicitation in response to mustard aphid Lipaphis erysimi (Kalt.) and the defense elicitor methyl jasmonate (MeJ). Identification of jas-monate-induced unigenes in B. juncea revealed that most are orthologous to aphid-responsive genes, identified in tax-onomically diverse plant–aphid interactions. the unigenes largely represented genes related to signal transduction, response to biotic and abiotic stimuli and homeostasis of reactive oxygen species (rOS), in addition to genes related to cellular and metabolic processes involved in cell organi-zation, biogenesis, and development. gene expression stud-ies revealed induction of the key jasmonate biosynthetic genes (LOX, AOC, 12-OPDR), redox genes (CAT3 and GST6), and other downstream defense genes (PAL, ELI3, MYR, and TPI) by several folds, both in response to MeJ and plant-wounding. However, interestingly aphid infesta-tion even after 24 h did not elicit any activation of these

Electronic supplementary material the online version of this article (doi:10.1007/s00425-014-2073-7) contains supplementary material, which is available to authorized users.

M. K. Koramutla · a. Kaur · M. negi · r. Bhattacharya (*) national research centre on Plant Biotechnology, Indian agricultural research Institute campus, new Delhi 110 012, Indiae-mail: rcb@nrcpb.org; rcbhattacharya1@gmail.com

P. Venkatachalam Department of Biotechnology, Periyar University, Salem 636 011, tamil nadu, India

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aliphatic glucosinolates, benzoxazinoid derivatives, etc. have been implicated in quantitative resistance to aphids, their genetics in defensive make up is unknown in culti-vated crop species including rapeseed-mustard (Halkier and gershenzon 2006; Meihls et al. 2013). Screening for naturally occurring resistance to aphids led to the identi-fication of large number of resistant accessions or unim-proved land races in several crops (reviewed in Dogimont et al. 2010). However, only a few major aphid-resistant r genes have been discovered so far. a landmark example of r gene is the Mi-1.2 gene primarily identified in wild tomato, Lycopersicon peruvianum (l.) P. Mill., which con-fers resistance to three species of the root knot nematode Meloidogyne (Milligan et al. 1998). the Mi-1.2 gene con-fers 100 % mortality to potato aphid Macrosiphum euphor-biae thomas (rossi et al. 1998) and resistance towards psyllids and whiteflies (nombela et al. 2003; casteel et al. 2006). Unlike Mi-1 gene the virus aphid transmission (Vat) gene from melon, Cucumis melo l., reduces fecundity of melon-cotton aphids Aphis gossypii glover by 80–90 % within 3 days (Klingler et al. 1998). In wheat, a Pto-like serine/threonine kinase gene and a Pti1-like kinase gene are up regulated in aphid Diuraphis noxia Mordvilko resist-ant plants (Boyko et al. 2006). Many of the r genes either encode nucleotide-binding site–leucine-rich repeat (nBS-lrr) type proteins or show tight linkages with nBS-lrr resistance genes (lagudah et al. 1997; Seah et al. 1998; Klingler et al. 2005).

In Brassica spp., despite a large number of attempts in the past, source germplasm for aphid resistance genes largely remain unavailable (Sekhon and ahman 1993; Bhadoria et al. 1995). In a relatively recent report, a wild crucifer, Brassica fruticulosa exhibited strong antibio-sis against mustard aphids (Lipaphis erysimi Kalt.) under laboratory-based screening. However, immediate attempt to introgress the antibiosis factors through B. juncea–fru-ticulosa introgression lines remained difficult due to com-plex and elaborate breeding requirements (atri et al. 2012). to overcome the bottleneck of resistance-source several attempts to develop aphid-resistant transgenic mustard also did not yield much success (Kanrar et al. 2002; Hossain et al. 2006). In the absence of specific r genes, attempts to identify up regulated transcripts in response to taxonomi-cally diverse plant–aphid interaction led to the identifica-tion of many orthologous transcripts. these transcripts encode proteins functioning in general plant defense and signaling, generation of reactive oxygen species (rOS), hypersensitive response, cell wall degradation, cell main-tenance, photosynthesis, and energy production (Boyko et al. 2006; Kempema et al. 2007). From the examples of major aphid-resistant genes and up regulated transcripts, it appears that a large number of plants’ innate immunity genes are involved in addition to specific gene-for-gene

recognition in aphid resistance (Smith and Boyko 2007). It seems likely that mechanistic differences in early sign-aling and activation processes of innate defense responses may account for the difference in quantitative resistance between the resistant and susceptible accessions.

among the key regulators of defense responses in plants, jasmonate-mediated signaling is primarily impli-cated to regulate antiherbivore defense (Halitschke and Baldwin 2004). Jasmonates are synthesized in plants via the octadecanoid pathway (creelman and Mullet 1997). In synthesizing jasmonates, lipoxygenase (lOX) oxygenates membrane-liberated linolenic acid, before it is converted to 12–oxo-phytodienoic acid (12-OPDa) by allene oxide synthase and allene oxide cyclase (aOc). reduction of 12-OPDa followed by three cycles of β-oxidation produces jasmonic acid (Ja) (Wasternack 2007). additional modi-fication of Ja leads to the formation of methyl jasmonate (MeJ) and its numerous conjugates collectively known as jasmonates. Octadecanoid-derived signals including MeJ play an important role in mounting host defense responses to herbivores mediated by defense proteins such as lectins, protease inhibitors (PIs), and polyphenol oxidases (Farmer and ryan 1992; rohwer and erwin 2010).

MeJ has been frequently used to elicit defense signaling against the chewing type of insect-pests (rohwer and erwin 2010; tian et al. 2014). However, studies demonstrating its effect on sap-sucking insects are rather limited. In arabidop-sis, constitutive expression of Ja in the cev1 mutant or exog-enous application of MeJ on cev1 as well as wild-type plants reduced multiplication of green peach aphids (Myzus per-sicae) (ellis et al. 2002). In tomato, MeJ-mediated defense elicitation produced a similar retarding effect on growth and fecundity of M. persicae populations (Boughton et al. 2006). the treated plants had increased levels of leaf peroxidases and polyphenol oxidase, indicative of induced host defense. though these reports emphasized the significance of endoge-nous defense proteins in conferring aphid antibiosis, they did not provide an accounting of the more intriguing question: how did the defense signaling in response to exogenous elic-itors differ from that, putatively elicited by aphid infestation. In cases of proven mechanistic or quantitative differences between the two, it may be hypothesized that it is the sign-aling and elicitation of jasmonate-mediated host defenses in perceiving aphid attack that differs among the plant types and contributes to genetic variance for resistance trait. Here, we show an attenuated expression pattern of host defense responses spanning the early and late defense genes in Indian mustard [B. juncea (l.) czern.] plants in response to mustard aphids (L. erysimi) and prove that its counteractive elicitation by MeJ-application leads to quantitative resistance to L. erysimi. Our results contribute to the understanding of plant–aphid interaction and the basis of aphid susceptibility in rapeseed-mustard.

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Materials and methods

Plant material and growth conditions

Seeds of B. juncea Bio-YSr were obtained from nrc on Plant Biotechnology, new Delhi, India. the plants were raised in plastic pots of 23-cm-diameter filled with steri-lized soilrite and maintained in a glass house under 16 h light (140 μmol m−2 s−1), 8 h dark cycles at 22 ± 2 °c, and 62–72 % relative humidity. the plants were irrigated with Hoagland’s nutrient solution twice a week. all the experi-ments were carried out using four-week-old healthy plants.

Insect rearing and insect inoculation

a colony of mustard aphid, L. erysimi, was maintained on B. juncea plants in isolation cages in the glasshouse as described above. to maintain the insect population, 3-week-old plants were freshly inoculated with aphids at one month intervals. For insect treatment, 100 apterae, adults of L. erysimi, were released on several individual 4-week-old B. juncea plants, allowed to settle and multiply. rna samples were collected from the infested plants at different time intervals from 0 to 24 h, after release of the aphids. It was ensured that only one sample was collected from any individual plant and for each time point samples from three different plants were pooled.

MeJ treatment and mechanical wounding of plants

Four-week-old plants with four to six expanded leaves were transferred to a growth chamber at least 1 day–night cycle before any treatment for stabilization. For MeJ treatment the plants were sprayed with a 100 µM solution of MeJ prepared in double-distilled water containing 0.1 % triton X-100. the leaf samples were collected at 0, 1, 3, 5, 8, and 24 h after spraying, immediately frozen in liquid nitrogen, and kept at −80 °c until used. For wounding, a hemostat was used to wound repeatedly across the mid-vein of the fifth and sixth leaves from the top. Unwounded systemic upper leaves from the wounded plants were collected at different time points as described above, for time course experiments. corresponding leaves from the unwounded plants served as controls for each time point. the leaf sam-ples were frozen in liquid nitrogen and kept at −80 °c until used.

Preparation of poly(a)+ rna and construction of a subtracted cDna library

For isolation of total rna, leaf tissues pooled from three different plants were ground to fine powder in liquid nitro-gen and transferred to trIzol (Invitrogen). rna was

isolated according to the manufacturer’s instructions. the typical yield of total rna was 50–80 µg per 100 mg leaf tissue. Poly(a)+ rna was purified from total rna using Magnetic mrna Isolation Kit (new england Biolabs).

Suppression subtractive hybridization (SSH) was car-ried out using the Pcr-Select™ Subtraction Kit (clon-tech). cDna prepared from MeJ-treated samples and water-treated control plants were used as the tester and the driver, respectively, in forward subtraction. the steps of subtraction were followed as described in the manual of the kit. the subtraction efficiency was evaluated by Pcr amplification of the housekeeping gene actin (acc. no. aF111812.1) in subtracted and unsubtracted cDnas. For amplification of 12-OPDA reductase (12-OPDr) a primer pair was developed based on its sequence (acc. no. gU085236.1) in B. juncea. the subtracted cDna was ligated to ta cloning vector pcr2.1 and transformed into Escherichia coli tOP10 competent cells using tOPO clon-ing kit (Invitrogen). White colonies were randomly picked and stored at −80 °c. the colonies were subjected to Pcr using M13 primers to confirm the presence and aver-age size of the inserts. Pcr reactions were carried out by amplifying 1 µg plasmid Dna in a cocktail of 25 µl con-taining 0.5 units taKara Taq (takara Bio Inc.) in 10× Pcr buffer with 1.5 mM Mg2+, 200 μM each dntP, and 0.4 μM each primer, for 25–28 cycles.

Southern hybridization of cDna macro array blot

cDna macro array analysis was performed according to Pcr-Select differential screening kit (clontech). Bacte-rial clones were grown overnight in lB media containing 50 μg ml−1 kanamycin. Following plasmid isolation, SSH inserts were Pcr amplified using adapter-specific prim-ers. Pcr amplicons were denatured with 0.6 M naOH at 37 °c for 15 min, blotted in duplicates onto Hybond-n membranes (ge Healthcare), neutralized in 0.5 M tris–Hcl (pH 7.5) for 3 min, and washed with double-distilled water. Dried blots were cross-linked by exposure to a UV dose of 120,000 µJ cm−2 using a UV cross linker. Hybridi-zation was performed according to manufacturer’s protocol and the membranes were washed in 2× SSc and 0.5 % SDS for 20 min followed by two washes in 0.2× SSc and 0.5 % SDS for 20 min each at 68 °c. the membranes were exposed to X-ray film (Kodak Biomax Mr Films) with an intensifying screen and kept at −80 °c for 48 h before developing.

nucleotide sequencing and data analysis

Dna sequencing was carried out by chromous Bio-tech, Bengaluru, India, using vector bound M13 primers. Sequence data were trimmed using ncBI VecScreen and

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assembled using the software DnaStar (DnaStar Inc., Madison, USa). Dna sequences were analyzed using the BlastX program of ncBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) at a threshold E value of 10−5 or better. the eSts were grouped into functional categories using the gO slim terms from the Arabidopsis information resource annota-tion (http://www.arabidopsis.org/tools/bulk/go/index.jsp) and Blast2gO software (götz et al. 2008).

Semi-quantitative rt-Pcr analysis

rna samples were treated with Dnase I (Invitrogen) and purified prior to semi-quantitative rt-Pcr. the rna (5 µg) was reverse transcribed in a 20-µl reaction volume using the Superscript III First-Strand cDna Synthesis Kit (Invitrogen) as per kit specification. to perform Pcr, 2 µl of cDna was amplified in a reaction cocktail for 25–28 cycles as described earlier. amplification of actin cDna (acc. no. aF111812.1) was used as an internal control to ensure equal amounts of cDna in each reaction tube. a list of the primer sequences used and the optimum num-ber of cycles to ensure linear amplification of the target genes are provided in Supplemental table S1 and Fig. S1, respectively. each reaction product was analyzed electro-phoretically on a 2 % agarose gel premixed with ethidium bromide. capture of high-resolution image and densiomet-ric analysis of the bands were performed in Bio-Imaging System using geneSnaP software version 6.00.26 (Syn-gene, MD, USa). the band intensity of each transcript was quantified using genetOOlS analysis software version 3.02.00 (Syngene, MD, USa). Data were normalized with the measured band intensity of actin for the same sample.

aphid bioassay on MeJ-elicited plants

Four-week-old plants, sprayed with MeJ as described ear-lier, were moved to a different chamber in the green house with similar growth conditions and left overnight to dry. On each plant ten apterae adults of L. erysimi were released with the help of a small paint brush. the inoculated plants were covered with thin box of transparent Plexiglas. For aeration the side walls of the boxes contained holes which were covered with cotton balls to prevent the escape of aphids. the increase in aphid population was recorded with the aid of a magnifying glass. Data were collected from three independent experiments with four replicates each. Means were compared within the treatments by anOVa and between the treatments by two-way anOVa.

Preparation of protein extracts and enzyme assays

leaf tissue (1 g) was homogenized in 10 ml of extraction buffer in a pre-chilled mortar and pestle. For cat, SOD,

and POD 0.2 M potassium phosphate buffer (pH 7.8) with 0.1 mM eDta was used as extraction buffer; whereas for aPX the extraction buffer was supplemented with 1 mM ascorbic acid. For PPO, 20 mM Hepes buffer (pH 7.2) and for phenyl ammonium lyase (Pal) 0.1 M sodium borate buffer (pH 8.8) with 20 mM β-mercaptoethanol were used. the homogenates were centrifuged at 15,000g, for 30 min at 4 °c for aPX, cat, SOD, and POD; at 10,000g for 20 min at 4 °c for PPO; and at 15,000g, 20 min at 4 °c for Pal to purify the supernatant. all enzyme extracts con-tained 5 % PVP, 1 % protease inhibitor cocktail for plant cell and tissue extracts (Sigma-aldrich). the supernatant was used for analysis of total protein content and spectro-photometric estimation of enzyme activity using evolu-tion 300 UV–Vis Spectrophotometer (thermo Scientific). the protein concentration was determined according to the method of Bradford (1976) using BSa as standard. the enzymes were assayed as follows:

SOD (ec 1.15.1.1): SOD activity was determined using a modified nitrobluetetrazolium (nBt) method as described by Beyer and Fridovich (1987). the assay was performed at room temperature in a 2-ml cuvette containing 50 mM phosphate buffer (pH 7.8), 2 mM eDta, 9.9 mM l-methio-nine, 55 µM nBt, 0.025 % triton-X100, 20 µl of 1 mM riboflavin, and 40 µl of enzyme extract. the reaction was initiated by illuminating samples under 15 W fluorescent tubes. One unit of SOD activity was defined as the amount of enzyme that inhibited the rate of nBt reduction by 50 % as monitored at 560 nm, under assay conditions.

cat (ec 1.11.1.6): cat activity was determined according to aebi (1984). the assay mixture (3 ml) con-tained leaf extract (2 ml; diluted 200 times in 50 mM potassium phosphate buffer, pH 7.0) and H2O2 (10 mM). cat activity was estimated by the decrease in absorbance of H2O2 at 240 nm and the specific activity was deter-mined using the molar absorptivity of hydrogen peroxide (40 mM−1 cm−1 at 240 nm). One unit of cat was defined as the amount of enzyme dismuting 1 µmol of hydrogen peroxide per min.

aPX (ec 1.11.1.11): aPX activity was carried out according to the modified method of nakano and asada (1981) in 3 ml of reaction mixture containing 50 mM potas-sium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.5 mM H2O2, and 10 µl of crude leaf extract. Oxidation of ascor-bate was determined by monitoring the decrease in absorb-ance at 290 nm (extinction coefficient 2.8 mM−1 cm−1). enzyme activity was expressed as units per mg of protein. One unit of aPX was defined as the amount of enzyme oxi-dizing 1 µmol of ascorbate per min.

POD: peroxidase activity was determined according to castillo et al. (1984). the reaction mixture (3 ml) contain-ing 50 mM phosphate buffer (pH 6.1), 16 mM guaiacol, and 100 µl of enzyme extract was prepared. the reaction

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was initiated by adding 0.5 ml of 12 mM H2O2 (final con-centration 2 mM) and change in absorbance was measured at 470 nm. the specific activity of peroxidase was deter-mined using the molar absorptivity of guaiacol at 470 nm (26.6 mM−1 cm−1) and expressed as µmol tetra-guaiacol formed per min per mg protein.

PPO (ec 1.10.3.2, ec 1.10.3.1, and ec 1.14.18.1): polyphenol oxidase activity was measured according to the modified method of Hori et al. (1997). the assay mix-ture consisted of 200 µl of crude enzyme extract, 200 µl of 0.2 M Hepes buffer (pH 6.0), 1 ml of 1.6 % catechol poly-phenol, and 600 µl of deionized water. the rate of increase in absorbance was measured at 420 nm for 1 min and the activity was expressed as Δa470 min−1 mg−1 protein.

Pal (ec 4.3.1.5): Pal activity was determined as the rate of conversion of l-phenylalanine to trans-cinnamic acid at 290 nm as described by Dickerson et al. (1984). the reaction mixture contained 0.1 ml extract, 3.9 ml of 0.01 mM sodium borate buffer, and 1 ml of 0.6 mM l-phe-nylalanine. the extract was replaced by 0.1 ml sodium borate buffer in control samples. the reactions were incubated at 37 °c for 1 h and stopped by the addition of 0.2 ml 6 M trichloroacetic acid. the specific activity of Pal was calculated using the molar extinction coefficient (9,630 mM−1 cm−1) and expressed as nmol cinnamic acid per min per mg protein.

Statistical analysis

the data were analyzed by graph pad prism software. the mean was derived from values of 2–3 biological replicates

with 1–4 technical replicates each (n = 2–3). comparison of means was carried out by student’s t test (P < 0.05).

Results

MeJ-induced transcriptomes in B. juncea and pathway classification

In rapeseed-mustard, information on genome sequences is limited and microarray chips are not available for transcript profiling. therefore, SSH technique was used for the iden-tification of jasmonate-induced transcripts. a subtractive cDna forward library representing MeJ-induced genes was constructed by taking cDna from MeJ-treated leaves and analogously water-treated leaves as ‘tester’ and ‘driver’, respectively. the efficiency of subtraction was evaluated by Pcr amplification of actin and the jasmonate induc-ible gene 12-OPDR. reduced abundance of actin mrna in subtracted samples compared to its initial abundance in unsubtracted sample indicated a high level of normali-zation and efficient subtraction (Fig. 1a). Pcr amplifica-tion of 12-OPDR transcripts appeared by the 18th cycle in subtracted (enriched) samples, whereas in unsubtracted samples the amplicon was visible only after 10 additional cycles. Subtracted cDnas were cloned into a Pcr vec-tor and 960 recombinant clones were identified. even after efficient subtraction, the tester sample might contain some of the cDnas, which failed to bind their counterpart in the driver sample and thus represent false positives. to minimize the frequency of false positives, 400 clones were

Fig. 1 Subtraction efficiency and enrichment of jasmonate-responsive cDnas in the SSH library of Brassica juncea leaves. a the subtracted and unsubtracted cDnas were amplified with the gene-specific primers for actin and 12-OPDR. aliquots of Pcr cocktails were taken after 18, 23, 28, 33 cycles of Pcr amplification and the products were analyzed on 2 % agarose gel. b a cDna macroarray of differentially expressed unigenes of B. juncea in response to MeJ. Pcr ampli-fied inserts from selected SSH clones were loaded on Hybond n membrane in duplicates and the membranes were hybrid-ized with radiolabeled cDna as probe either from water-treated (D) or MeJ-treated (T) plants

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analyzed by macroarray (dot blot) analysis using cDnas from MeJ-treated samples as test probes and cDnas from water-treated samples as driver probes (Fig. 1b). a total of 152 clones were identified from the subtracted library showing intense hybridization to the test probe and weak hybridization to the driver probe.

the cDna inserts of 152 identified clones, fil-tered through VecScreen to remove any terminal vector sequences, were assembled using DnaStar software for the identification of unique eSts. a total of 135 uni-genes were obtained out of 152 sequence reads that indi-cated a very low level of redundancy in the SSH library. the unigenes were analyzed by the ncBI BlastX program for identification of their homologous genes in the eMBl/genBank databases and e-values of the matches (table 1). Based on the top matches and their putative function in biological processes, all of the unigenes were categorized into various functional groups. among 135 unigenes, 33 (24 %) did not show any match either to any cDna or pro-tein sequences in the database and were included in the cat-egory of ‘novel’. the other unigenes (76 %) displayed high similarities to plant genes with known and unknown func-tions. these 102 unigenes were categorized into 13 groups as listed in Fig. 2. In a functional classification based on Blast2go analysis, 102 unigenes were assigned to one or more gO terms. these unigenes were categorized into three gO ontologies viz. biological processes, molecular function, and cellular components (Fig. 2). In biological processes, the biggest group was formed by genes involved in cell processes (30 %). this was followed by genes related to response to stimuli (24 %) (Fig. 2b). In the cel-lular component category, 42 % of the genes were confined to cell followed by organelle (32 %) (Fig. 2c). In molecular functions, 45 % of the genes were found to have catalytic activity and 42 % had binding activity (Fig. 2d).

expression analysis of defense genes in B. juncea in response to aphid infestation

to study the gene expression of jasmonate-mediated host defense against aphids in B. juncea, we narrowed down the candidates to ten unigenes consisting of three octadeca-noid pathway genes directly involved in MeJ metabolism, two stress-responsive redox genes and four downstream insect defense genes (table 2) for expression analysis. In insect-inoculated plants, most of the aphids started pro-boscis within 2 h of release as indicated by immobiliza-tion of the individuals at the site of feeding. time course experiments on gene expression indicated that initial pro-boscis and colonization by aphids could not elicit activa-tion of any of the three octadecanoid pathway genes, even after 24 h of aphid release (Fig. 3). However, similar time course experiments on the MeJ-treated and mechanically

wounded plants indicated transcriptional activation of all three genes in both the treatments with differential activa-tion patterns. the activation of LOX and 12-OPDR was greater in the case of MeJ treatment, with maximas of 7.5-fold at 5 h and 5.9-fold at 3 h, respectively, compared to their wound responses. In contrast, the activation of AOC was more profound in the case of mechanical wounding, with a maximal increase of 26.6-fold in transcript level at 5 h post-wounding compared to an eightfold increase for MeJ treatment.

Hydrogen peroxide (H2O2) is an important signal mol-ecule in jasmonate-mediated herbivore defense (Orozco-cardenas et al. 2001). two alternative redox genes viz. catalase (CAT) and glutathione S-transferase (GST) are associated with H2O2 metabolism in MeJ signaling. We analyzed the activation pattern of these two redox genes in response to aphid attack in parallel with MeJ treatment and mechanical wounding. time course expression pat-tern of CAT3 showed transcript induction for all the three treatments (Fig. 4). Interestingly, in aphid-infested plants induction of CAT3 transcription began at 1 h and the tran-script level reached a maximum at 5 h, with approximately a fivefold increase compared to the initial level at 0 h. For MeJ-treated and wounded plants, expression of CAT3 increased to 3- and 3.1-fold of control levels at 1 and 5 h, respectively. the transcript level of GST6 in aphid-colo-nized plants remained similar to uninfested control plants 24 h after aphid inoculation. In contrast, GST6 transcrip-tion was activated by MeJ and wound treatment with maxi-mal induction levels of 7.5- and 12.4-fold increase at 1 and 3 h, respectively. the GST6 transcripts continued to remain high even after 24 h of treatment.

to ascertain if downstream defense genes are activated in response to aphid attack in B. juncea plants, transcript levels of genes encoding Pal, elicitor responsive gene (elI3), myrosinase (MYr) and trypsin protease inhibitor (tPI) were assayed in time course experiments following aphid inoculation. Interestingly, all four genes displayed no significant change in transcripts levels in response to aphid infestation in B. juncea plants even after 24 h of insect inoculation. However, all of the genes demonstrated significant activation of transcript levels in response to MeJ application and plant wounding (Fig. 5). In MeJ-treated plants, PAL transcript level gradually increased to 2.3-fold of initial time point levels up to 5 h, followed by a further increase beyond 8 h that remained high even after 24 h. In wounded plants, PAL transcript level rap-idly peaked at 1 h by 11-fold increase. In response to MeJ treatment as well as wounding, ELI3 and MYR transcript activation showed a similar pattern, both peaking at 5 h. In the case of TPI, there was a sharp increase in transcript level due to MeJ treatment and wounding which started declining after 3 h.

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Table 1 Major up regulated unigenes in Brassica juncea in response to MeJ with putative and unknown function

genbank accession no. Putative function Organism E value

response to stress

JZ482656 alpha-glucan water dikinase 1 (SeX1) Arabidopsis thaliana 2e−32

JZ482537 Hypothetical protein Arabidopsis thaliana 5e−30

JZ482531 Opc-8:0 coa ligase1 Arabidopsis lyrata 8e−32

JZ482558 Iaa-amino acid hydrolase 3 Brassica rapa 2e-26

JZ482542 Myrosinase-binding protein Brassica rapa 1e−49

JZ482577 Hypothetical protein BBa_01712 Beauveria bassiana 2e−05

JZ482563 12-oxophytodienoate reductase 1 Arabidopsis lyrata 4e−51

JZ482589 12-oxo-phytodienoate reductase 3 Arabidopsis thaliana 2e−07

JZ482608 Maternal effect embryo arrest 14 protein Arabidopsis thaliana 7e−67

JZ482547 Desiccation responsive protein Arabidopsis thaliana 1e−20

JZ482582 glutathione S-transferase (gSt6) Arabidopsis thaliana 3e−07

JZ482639 atP-dependent zinc metalloprotease FtSH 2 Arabidopsis thaliana 8e−63

JZ482579 aquaporin (plasma membrane intrinsic protein 2c) Arabidopsis thaliana 2e−18

JZ482654 erD15 protein Brassica napus 7e−52

lIBeSt_028273 Phenylalanine ammonia-lyase Brassica rapa 9e−27

JZ482560 rapeseed putative trypsin inhibitor 1 Brassica napus 2e−34

JZ482583 glutathione transferase Brassica juncea 4e−47

JZ482535 Putative branched-chain-amino-acid aminotransferase 4 Brassica rapa 1e−54

JZ482543 allene oxide cyclase 2 Arabidopsis thaliana 4e−24

JZ482532 elI3 (pyridine nucleotide-disulfide oxidoreductase family protein) Arabidopsis lyrata 2e−62

JZ482638 lipoxygenase Brassica oleracea 2e−30

lIBeSt_028273 catalase 3 Brassica rapa 2e−59

JZ482544 Xyloglucosyl transferase 1, partial Brassica juncea 7e−79

JZ482546 auxin-responsive gH3 family protein Arabidopsis thaliana 2e−46

JZ482632 Beta-amylase 8 Arabidopsis thaliana 1e−22

JZ482536 cytochrome P450 83B1, partial Brassica oleracea 7e−24

JZ482539 WrKY Dna-binding protein 18 Arabidopsis thaliana 8e−31

JZ482590 chlorophyll a/b-binding protein cP29 Arabidopsis thaliana 2e−62

JZ482545 naDP-dependent malic enzyme 3 Arabidopsis thaliana 1e−50

JZ482575 Myrosinase, thioglucoside glucohydrolase Brassica juncea 8e−45

JZ482591 Putative hydroperoxide lyase HPOl Arabidopsis thaliana 2e−55

JZ482596 anthranilate N-benzoyltransferase Arabidopsis thaliana 5e−43

JZ482637 Heat shock protein 70 Arabidopsis thaliana 6e−75

JZ482623 ribulose bisphosphate carboxylase small chain 1B Arabidopsis thaliana 9e−04

JZ482554 Defense-related protein Brassica carinata 7e−27

JZ482602 Pgr5-like protein 1a Arabidopsis thaliana 1e−20

JZ482645 Zinc-dependent protease Arabidopsis thaliana 2e−33

Protein metabolism

JZ482581 epsilon-adaptin, putative Arabidopsis thaliana 4e−14

JZ482585 60S ribosomal protein l18a Medicago truncatula 7e−15

JZ482566 leucine-rich repeat transmembrane protein kinase Arabidopsis thaliana 5e−37

JZ482666 aspartyl protease family protein Arabidopsis lyrata 2e−09

JZ482601 chaperone protein dnaJ-like protein Arabidopsis thaliana 6e−39

JZ482621 rna polymerase beta subunit Brassica napus 2e−36

JZ482622 ribosomal protein S12 Cynomorium songaricum 7e−36

JZ482598 Putative UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltrans-ferase Sec

Arabidopsis thaliana 7e−40

JZ482593 naDP-specific isocitrate dehydrogenase-like protein Arabidopsis thaliana 2e−19

184 Planta (2014) 240:177–194

1 3

Table 1 continued

genbank accession no. Putative function Organism E value

JZ482635 Peptidyl-prolyl cis–trans isomerase FKBP12-like Cucumis sativus 8e−27

Signal transduction

JZ482540 Protein tIFY 10a Arabidopsis thaliana 2e−09

JZ482574 Protein tIFY 11B Arabidopsis thaliana 1e−21

JZ482587 Protein tIFY 11a Arabidopsis thaliana 8e−22

JZ482552 Sulfotransferase 5a Brassica rapa 1e−65

JZ482586 WrKY40-1 transcription factor Brassica napus 1e−10

JZ482562 cytochrome P450, family 94, subfamily c, polypeptide 1 Arabidopsis thaliana 1e−41

JZ482568 aMP deaminase Arabidopsis thaliana 8e−102

JZ482619 Malate dehydrogenase 2 Brassica napus 2e−45

JZ482564 Mtn19-like protein Arabidopsis thaliana 4e−36

JZ482600 gF14 omega Brassica napus 1e−49

JZ482604 c2H2 type zinc finger protein Brassica rapa 1e−48

transport

JZ482569 atPDr7/PDr7 Arabidopsis lyrata 1e−17

JZ482567 chloroplast envelope ca2+-atPase precursor Arabidopsis thaliana 9e−99

JZ482549 PDr8/Pen3 Arabidopsis lyrata 2e−17

JZ482556 atPase e1–e2 type family protein/haloacid dehalogenase-like hydrolase family protein

Arabidopsis thaliana 2e−27

JZ482573 Plant synaptotagmin Arabidopsis thaliana 3e−23

JZ482588 Vacuolar-type H+-atPase subunit a Arabidopsis thaliana 5e−24

JZ482640 transketolase-like protein Arabidopsis thaliana 3e−12

JZ482594 Similar to cgI-126 protein Arabidopsis thaliana 5e−14

JZ482633 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein

Arabidopsis thaliana 1e−21

JZ482561 PDr5-like aBc transporter from Spirodela polyrrhiza 2e−38

cell organization and biogenesis

JZ482641 Uncharacterized protein Arabidopsis thaliana 8e−60

JZ482571 Pectinesterase 25 Arabidopsis thaliana 3e−18

JZ482576 aDP-glucose pyrophosphorylase large subunit Brassica rapa 1e−27

JZ482644 tubulin beta-9 chain Arabidopsis thaliana 1e−61

Developmental processes

JZ482550 topless-related 2 protein (tPr2) Arabidopsis thaliana 2e−14

JZ482541 Zinc-binding dehydrogenase family protein Arabidopsis thaliana 5e−72

transcription, Dna-dependent

JZ482572 Zinc finger (B-box type) family protein Arabidopsis lyrata 1e−04

JZ482533 rIng/FYVe/PHD zinc finger domain-containing protein Arabidopsis thaliana 4e−74

electron transport or energy pathways

JZ482615 chlorophyll a/b binding protein Brassica oleracea 2e−82

JZ482534 tPa: hypothetical protein ZeaMMB73_942389 Zea mays 2e−33

Unknown biological processes

JZ482642 nodulin Mtn21-like transporter family protein UMaMIt45 Arabidopsis thaliana 4e−60

JZ482651 Unknown Brassica rapa 3e−72

JZ482595 Hypothetical protein carUB_v10026553 mg Capsella rubella 3e−30

JZ482616 OrF 143 Glycine max 3e−13

JZ482592 Dna-binding protein Arabidopsis thaliana 4e−41

Other biological processes

JZ482646 Putative retroelement pol polyprotein Arabidopsis thaliana 2e−33

JZ482570 nHP2 non-histone chromosome protein 2-like 1 Danio rerio 2e−27

185Planta (2014) 240:177–194

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attenuation of the aphid population on MeJ-treated plants

the extent of quantitative resistance to mustard aphids in MeJ-treated B. juncea plants was assayed by recording the growth of an aphid population on the treated plants over a period of 7 days (Fig. 6). In MeJ-treated plants, although there was a significant increase in the aphid population at 3 days post-inoculation, the rate of parthenogenetic mul-tiplication was arrested at later time points as indicated by nonsignificant differences in mean-aphid population recorded after 5 and 7 days (F4,10 = 9.02, P = 0.002) (Fig. 6a). In treated plants, the total number of aphids increased by only sevenfold in 7 days post-inoculation. In contrast, with control plants, analogously treated with water, the aphid population rapidly increased threefold within 24 h of inoculation and further increased to 29-fold within 7 days of inoculation (F4,10 = 16.20, P = 0.0002). group comparison between the treatments and a two-way anOVa indicated a significant retarding effect of MeJ on the multiplication of aphids (F1,4 = 66.91, P = 0.0012). also, the mean aphid-biomass per plant was significantly less in the MeJ-treated plants compared to the control plants (Fig. 6b). the results demonstrated antibiosis effects of MeJ-treated plants on the growth and reproduction of aphids. However, no significant insect-mortality was observed either in the MeJ treated or in the control plants.

antioxidant defense in B. juncea in response to MeJ treatment

as the common denominator of insect and pathogen defense responses, plants activate major enzymatic anti-oxidants viz. ascorbate peroxidase (aPX), superoxide dis-mutase (SOD), and catalase (cat), which are involved in scavenging rOS, generated as a result of plant–insect/pathogen interactions (Karpinski and Muhlenbock 2007; lee et al. 2007). these redox enzymes are used as mark-ers of host defense signaling (Bhattacharya et al. 2013). We compared the level of antioxidant enzymes cat, aPX, and SOD in leaf-homogenates of the aphid-infested plants which were treated either with MeJ or water (Fig. 7a). Uni-noculated healthy plants analogously treated with water were used as the control. Interestingly, aphid infestation per se did not evoke any activation of leaf-cat, -aPX or-SOD activity, which remained at a similar level to the uninfested control plants with the exception of a transient, moderate increase in cat activity at 3 days period in aphid-infested plants. In contrast, MeJ treatment significantly increased the specific activity of all three antioxidant enzymes com-pared to water-treated control plants. However, the quan-titative maxima and the time course pattern of activation differed among the individual enzymes. Following the MeJ treatment, aPX activity gradually increased from 1 day

Table 1 continued

genbank accession no. Putative function Organism E value

JZ482597 chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit precursor

Brassica napus 3e−58

JZ482650 Ycf2 Pachycladon cheesemanii 3e−11

Other cellular processes

JZ482653 Ferredoxin thioredoxin reductase catalytic beta chain family protein Arabidopsis lyrata 4e−56

JZ482617 cytochrome P450 monooxygenase 83a1–5 Brassica napus 2e−14

JZ482578 aPS reductase Brassica juncea 5e−28

JZ482553 Water soluble chlorophyll protein Brassica oleracea 6e−30

JZ482548 alpha/beta-hydrolase domain-containing protein Arabidopsis thaliana 3e−16

JZ482559 cytochrome b5 Brassica oleracea 2e−15

JZ482663 nmra-like negative transcriptional regulator-like protein Arabidopsis thaliana 1e−44

JZ482555 naDPH-cytochrome P450 reductase 2 Arabidopsis thaliana 3e−29

Other metabolic processes

JZ482584 cytochrome P450, family 72, subfamily a, polypeptide 13 Arabidopsis thaliana 2e−51

JZ482557 FaD-binding domain-containing protein Arabidopsis lyrata 4e−47

JZ482538 HaD superfamily, subfamily IIIB acid phosphatase Arabidopsis thaliana 1e−12

JZ482565 cytochrome P450, family 715, subfamily a, polypeptide 1 Arabidopsis thaliana 6e−61

JZ482628 cytochrome P450 71B26 (cYP71B26) Arabidopsis thaliana 1e−96

JZ482599 atP-citrate lyase a-1 Arabidopsis thaliana 6e−52

JZ482551 Hypothetical protein carUB_v10024617 mg Capsella rubella 5e−31

novel unigenes with unknown function: JZ482664, JZ482580, JZ482603, JZ482607, JZ482609, JZ482610, JZ482611, JZ482612, JZ482613, JZ482614, JZ482618, JZ482620, JZ482624, JZ482625, JZ482626, JZ482627, JZ482629, JZ482630, JZ482634, JZ482636, JZ482643, JZ482647, JZ482648, JZ482649, JZ482652, JZ482655, JZ482657, JZ482658, JZ482659, JZ482660, JZ482661, JZ482662, JZ482665

186 Planta (2014) 240:177–194

1 3

through 5 days reaching a peak at 5 days, beyond which the basal level of activity was restored. the pattern of activa-tion for SOD and cat was similar and indicated that there was an immediate burst of activity of these two enzymes, reaching a maxima within 24 h of treatment and a gradual decrease thereafter until basal levels were restored at 7 days post-treatment. Variations observed in water-treated control plants were statistically insignificant, indicating that the increase in specific activity of the three enzymes was medi-ated by MeJ treatment.

elicitation of defense enzymes in response to MeJ treatment in B. juncea

Plant defense enzymes peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (Pal)

modulate levels of plant secondary metabolites and are involved in endogenous defense response of plants against different types of biotic stress (Han et al. 2009; tian et al. 2014). We compared the activity levels of POD, PPO, and Pal between the control aphid-infested and the MeJ-treated aphid-inoculated plants to ascertain whether these defense enzymes contribute to the antibiosis conferred by MeJ treat-ment in B. juncea plants. the specific activity of all three enzymes increased in a similar pattern after treatment with MeJ (Fig. 7b). In each case the time course of induction pattern indicated a gradual increase in the specific activity of each enzyme over a time period of 1–3 or 5 days post-treatment, reaching a maximal induction either at 3 days post-treatment, as in the case of POD and PPO or at 5 days post-treatment as shown by Pal. the maximum activation recorded in the case of POD and PPO was ~2- and 2.3-fold,

Fig. 2 Functional grouping of differentially expressed B. juncea uni-genes indentified from SSH forward library of MeJ-treated plants. a Based on gene ontology the up regulated genes were classified into 13 functional categories indicated by different letters in the pie chart. the functional categories are A other cellular processes, B other met-abolic processes, C response to abiotic or biotic stimuli, D response

to stress, E other biological processes, F transport; G signal transduc-tion, H cell organization and biogenesis, I developmental processes, J protein metabolism, K electron transport or energy pathway, L transcription, Dna-dependent, M unknown biological processes. b–d Percent distribution of gO terms: biological process (b), cellular component (c) and molecular function (d)

187Planta (2014) 240:177–194

1 3

Tabl

e 2

Sel

ecte

d ca

ndid

ate

gene

s fo

r ex

pres

sion

ana

lysi

s of

hos

t-de

fens

e re

spon

se to

L. e

rysi

mi i

n B

. jun

cea

gen

eFu

nctio

na

ctiv

ityr

efer

ence

s

LO

X (

lipox

ygen

ase)

Hom

olog

ous

to a

rabi

dops

is L

OX

2 (a

t3g4

5140

). a

dditi

on o

f th

e m

olec

ular

ox

ygen

to p

oly

unsa

tura

ted

fatty

aci

d ha

ving

ci

s, c

is-1

, 4-p

enta

dien

e to

yie

ld a

n un

satu

-ra

ted

fatty

aci

d w

ith h

ydro

pero

xide

Her

bivo

re, p

atho

gen,

and

wou

nd d

efen

se s

ign-

alin

g; s

eed

germ

inat

ion,

veg

etat

ive

grow

th

and

deve

lopm

ent i

n pl

ants

Port

a an

d r

ocha

-Sos

a (2

002)

, chr

iste

nsen

et a

l. (2

013)

AO

C (

alle

ne o

xide

cyc

lase

)H

omol

ogou

s to

ara

bido

psis

AO

C2

(at3

g257

70).

Ste

reos

peci

fic c

ycliz

atio

n of

th

e un

stab

le a

llene

oxi

de in

to th

e st

able

cis

-(+

) en

antio

mer

OPD

a

ear

ly d

efen

se g

ene

invo

lved

in ja

smon

ate

(Ja

)-m

edia

ted

defe

nse

resp

onse

; ind

uced

by

inse

ct, w

ound

ing

and

path

ogen

; im

pair

men

t of

aO

c in

ric

e le

ads

to m

ore

susc

eptib

ility

to

war

ds b

last

fun

gus

Sten

zel e

t al.

(200

3), a

be e

t al.

(200

8),

rie

man

n et

al.

(201

3)

12-O

PD

R (

12-o

xo-p

hyto

dien

oic

acid

red

uc-

tase

)c

atal

yzes

the

redu

ctio

n of

dou

ble-

bond

s in

α,

β-u

nsat

urat

ed a

ldeh

ydes

or

keto

nes

to y

ield

th

e co

rres

pond

ing

hexa

noic

aci

d de

riva

tives

Sign

al m

olec

ule

in J

a-m

edia

ted

defe

nse

resp

onse

; wou

nd r

espo

nse;

def

ense

res

pons

e to

whe

at a

phid

(D

. nox

ia);

pat

hoge

n de

fens

e; in

duce

d by

pla

nt h

orm

ones

MeJ

, Sa

, et

aB

a

taki

et a

l. (2

005)

, Mar

imut

hu a

nd S

mith

(20

12)

CA

T (

cata

lase

)D

ism

utat

ion

of to

xic

H2O

2 in

to w

ater

and

m

olec

ular

O2

cri

tical

in m

aint

aini

ng th

e re

dox

bala

nce

dur-

ing

oxid

ativ

e st

ress

due

to b

iotic

and

abi

otic

st

ress

es; a

ctiv

ated

by

defe

nse

sign

alin

g;

invo

lved

in p

lant

res

ista

nce

to in

sect

her

bi-

vore

s, b

acte

rial

and

fun

gal p

atho

gens

Mha

mdi

et a

l. (2

010)

, Bha

ttach

arya

et a

l. (2

013)

GST

(gl

utat

hion

e S-

tran

sfer

ase)

cat

alyz

e gl

utat

hion

e-de

pend

ent i

som

eriz

a-tio

ns a

nd r

educ

tion

of to

xic

orga

nic

hydr

op-

erox

ides

a m

arke

r fo

r pl

ant r

espo

nse

to s

tres

s; a

ct a

s si

gnal

ing

mol

ecul

es in

act

ivat

ing

phen

yl-

prop

anoi

d m

etab

olis

m; i

nvol

ved

in f

unga

l re

sist

ance

in to

bacc

o; in

duce

d by

mec

hani

-ca

l wou

ndin

g, c

hew

ing

and

sap

suck

ing

inse

cts

Stot

z et

al.

(200

0), D

ean

et a

l. (2

005)

, K

empe

ma

et a

l. (2

007)

PAL

(ph

enyl

alan

ine

amm

onia

lyas

e)c

atal

yzes

rat

e co

ntro

lling

ste

p of

phe

nylp

ro-

pano

id m

etab

olis

m: d

eam

inat

ion

of p

heny

la-

lani

ne to

pro

duce

tran

scin

nam

ic a

cid

Invo

lved

in p

heny

lpro

pano

id a

nd it

s br

anch

pa

thw

ays

lead

s to

the

synt

hesi

s of

div

erse

de

fens

e co

mpo

unds

viz

. lig

nin

and

sube

rin,

fu

rano

coum

arin

, pte

roca

rpan

, etc

., an

d si

gnal

mol

ecul

es s

uch

as S

a; i

nvol

ved

in

syst

emic

acq

uire

d re

sist

ance

in to

bacc

o

He

et a

l. (2

011)

EL

I3 (

elic

itor

resp

onsi

ve g

ene)

Der

ivat

ize

arom

atic

aci

d an

d al

dehy

des

to

defe

nse

rela

ted

arom

atic

alc

ohol

se

ncod

es a

rom

atic

alc

ohol

ic n

aD

P+ o

xido

re-

duct

ase;

def

ense

com

poun

d in

ara

bido

psis

, pa

rsle

y, M

edic

ago

and

pota

to; i

nduc

ed b

y pa

thog

ens

and

sign

al m

olec

ules

viz

. Sa

, et

hyle

ne, M

eJ

Som

ssic

h et

al.

(199

6), M

onte

sano

et a

l. (2

003)

MY

R (

myr

osin

ase)

Myr

osin

ase

clea

ves

the

thio

-lin

ked

gluc

ose

of

a cl

ass

of c

ompo

unds

cal

led

gluc

osin

olat

es

by h

ydro

lysi

s

act

ivat

ed b

y ja

smon

ate-

med

iate

d de

fens

e;

hydr

olyz

e gl

ucos

inol

ates

to p

rodu

ce to

xic

com

poun

ds li

ke is

othi

ocya

nate

s; m

ore

effe

c-tiv

e ag

ains

t gen

eral

ist i

nsec

ts

ras

k et

al.

(200

0), H

alki

er a

nd g

ersh

enzo

n (2

006)

TP

I (t

ryps

in p

rote

ase

inhi

bito

r)t

PI in

hibi

ts tr

ypsi

n en

zym

e by

for

min

g in

solu

ble

com

plex

try

psin

pro

teas

e in

hibi

tors

are

pla

nts

inna

te

defe

nse

prot

eins

indu

ced

agai

nst i

nsec

t he

rbiv

ory

leo

et a

l. (1

998)

, Zav

ala

et a

l. (2

004)

188 Planta (2014) 240:177–194

1 3

respectively, relative to basal level, whereas Pal activity increased to 4.7-fold of the basal level at its peak. How-ever, in aphid-infested plants the aphid-inflicted cues did not

elicit any activation of these defense enzymes and any vari-ation observed between the samples was statistically insig-nificant relative to the control.

Fig. 3 expression analysis of jasmonate biosynthetic genes in response to aphid infestation, MeJ treatment, and wounding. Four-week-old B. juncea plants were subjected to aphid inoculation, methyl jasmonate (+MeJ), and wounding (+Wnd) across the main vein with a hemostat followed by their incubation under light. total rna was isolated from the leaves at different time intervals of 1, 3,

5, 8 and 24 h and assayed for the expression of the jasmonate biosyn-thetic genes LOX, AOC and 12-OPDR by semi-quantitative rt-Pcr with actin as an internal control. the wounded (W) sample, showing the highest expression of the genes, was compared to the unwounded control (c) plants. Values represent mean ± Se (n = 3). Different let-ters indicate significantly different values

Fig. 4 expression analysis of defense-related redox genes in response to aphids, MeJ, and wounding. total rna col-lected from plants treated with aphids, MeJ and wounding were analyzed by semi-quantitative rt-Pcr using CAT3 and GST6 specific primers. amplification of actin was used as internal control. Fold change in expres-sion was derived based on integrated density values (IDV) of the amplicons run on 2 % agarose gel. Values represent mean ± Se (n = 3). Different letters indicate significantly dif-ferent values

189Planta (2014) 240:177–194

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Discussion

In many cultivated crops including rapeseed-mustard, quantitative resistance to aphids is limited (Sekhon and ahman 1993; Dogimont et al. 2010). the existing cul-tivars as well as wild relatives of Indian mustard do not show much genetic variability in terms of resistance and are especially susceptible to aphids (Bhadoria et al. 1995). Induced defense responses, when activated through exog-enous elicitor viz. MeJ, can restrict population growth and fecundity of the green peach aphid M. persicae, in suscep-tible plants of tomato and arabidopsis (ellis et al. 2002; Boughton et al. 2006). In Brassica species, it is not known

if the induced defense response elicited by exogenous MeJ can confer a similar aphid-retarding antibiosis and if the aphids or aphid-inflicted tissue damage can evoke a defense response.

In induced host defense against plant herbivores, jas-monate-mediated signaling is the major pathway. Jas-monate-responsive defense genes are commonly activated through application of MeJ (Baldwin 1998; li et al. 2002). to study the induction pattern of jasmonate-inducible defense genes against aphids in B. juncea, it was imperative to identify them due to the limited availability of genomics data on Brassica species in the public domain databases. In Brassica species, cDna microarrays for gene expression

Fig. 5 gene expression study of late defense genes in B. juncea in response to aphids, MeJ, and wounding. Four-week-old plants were subjected to aphid infestation, MeJ, and mechanical wounding. total rna was isolated at different time intervals. transcript levels of the

genes ELI3, MYR, and TPI were analyzed by semi-quantitative rt-Pcr using actin as internal control. Fold change in expression was derived as described earlier. Values represent mean ± Se (n = 3). Different letters indicate significantly different values

Fig. 6 analysis of population growth of L. erysimi on methyl jasmonate-treated B. juncea plants. Four-week-old mustard plants either treated with MeJ or water were infested with ten apterae adults of L. erysimi per plant. aphids were counted on 0, 1, 3, 5, and 7 days post inoculation. Values represent mean ± Se (n = 3). Asterisks in a and different letters in b indicate significant difference (P < 0.05) between the means

190 Planta (2014) 240:177–194

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profiling are not available. although arabidopsis microar-rays have been occasionally used in profiling B. napus gene expression, such attempts seemed impractical in B. juncea due to the larger genome size and more divergence of the latter from arabidopsis (carlsson et al. 2007; lee et al.

2008). to circumvent the unavailability of microarray chips in many of the cultivated crop species, SSH technique has been extensively used to selectively identify cDnas dif-ferentially expressed in the defense-elicited samples (Divol et al. 2005; Park et al. 2006; Boyko et al. 2006). the SSH

Fig. 7 Biochemical assay of plant defense enzymes in aphid-inocu-lated B. juncea plants. total proteins were extracted from the leaves of aphid-inoculated plants treated with either MeJ or water prior to insect release for estimation of the enzyme activities. Mean spe-cific activities (n = 2; ±Se) were determined in a time course man-

ner over a time period of 0–7 days and compared. a time course of specific activities of redox enzymes cat, aPX and SOD. b Specific activities of defense enzymes POD, PPO, and Pal determined in samples as described above. Different letters indicate significant dif-ference (P < 0.05)

191Planta (2014) 240:177–194

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cDna library of MeJ-treated B. juncea leaves represented eSts of jasmonate-activated genes in B. juncea. Selected clones were further verified in macroblot analysis for their true differential expression due to MeJ treatment, and screened to remove redundancy, if any, to identify the set of unigenes. the identified unigenes largely represented genes related to cellular and metabolic processes, genes respon-sive to biotic and abiotic stimuli, and homeostasis of rOS, in addition to genes involved in signal transduction, cell organization, biogenesis and developmental processes. a large proportion of the genes was similar and orthologous to the aphid-responsive genes identified in arabidopsis–M. persicae, nicotiana–M. nicotianae, Sorghum–Schizaphis graminum, and Wheat–D. noxia interaction (Smith and Boyko 2007). However, from the functional classification, it was difficult to signify the importance of any specific pathway in mounting the host defense response against aphids, since MeJ also regulate diverse developmental pro-cesses in plants (creelman and Mullet 1997).

the expression patterns of selected unigenes encod-ing different components of the jasmonate-mediated host defense were analyzed in a time course manner following aphid infestation in B. juncea. transcriptional activation of jasmonate biosynthetic genes is responsible for mediat-ing the intracellular jasmonate burst in the early signaling of herbivore defense in plants including caterpillar-resist-ant maize (Shivaji et al. 2010). lOX, aOc and 12-OPDr constitute three key enzymes in the jasmonate biosynthetic pathway. Interestingly, time course experiments on gene expression of aphid-infested B. juncea plants indicated that tissue infliction and feeding by L. erysimi did not elicit any transcriptional activation of octadecanoid pathway genes responsible for endogenous jasmonate generation. this would seem to indicate that B. juncea–L. erysimi interac-tion does not activate jasmonate-mediated signaling of herbivore defense. Unlike chewing insects, sap-sucking aphids minimize wound responses by limiting cell damage in feeding (guerrieri and Digilio 2008). In parallel experi-ments on MeJ treatment and mechanical wounding of B. juncea plants, profound transcriptional activation of LOX, AOC and 12-OPDR in MeJ-treated as well as wounded leaf samples support the hypothesis that the limited aphid-inflicted tissue damage failed to evoke any wound response to activate jasmonate biosynthetic genes.

the jasmonate signaling pathway activates naDPH oxi-dase to generate H2O2 as a secondary messenger to acti-vate downstream defense proteins (Orozco-cardenas et al. 2001). accumulation of H2O2 in response to aphids and its plausible role in defense signaling have been demonstrated in many plant species such as arabidopsis, wheat, bar-ley, etc. (argandoña et al. 2001; Moloi and van der West-huizen 2006; Kusnierczyk et al. 2008). Increased level of H2O2 is toxic to the host cells, and, therefore, the plants

concomitantly generate antioxidant defense enzymes to scavenge the toxic effects of H2O2. In chrysanthemum and triticale, the aphid-resistant cultivars had increased levels of antioxidant enzymes compared to the susceptible types (He et al. 2011; lukasik et al. 2012). In B. juncea, out of sev-eral isoforms of the redox genes catalase and GST, the SSH library data indicated maximum abundance for CAT3 and GST6. expression of CAT3 was more profoundly induced by MeJ application compared to wound treatment; wound treatment produced a greater induction on GST6 (Fig. 4). this might indicate a coordinated action in rOS homeosta-sis under jasmonate signaling and wound response. Imme-diately after aphid release, in B. juncea leaves, CAT3 dem-onstrated a gradual transcript accumulation which persisted 8 h after infestation. Higher catalase activity reduces endog-enous level of H2O2 which acts as a secondary messenger for defense signaling against herbivores. therefore, an early activation of catalase activity might be inhibitory to defense signaling that activate downstream defense genes in B. jun-cea plants. genes encoding downstream defense proteins viz. ELI3, MYR, and TPI showed prominent up-regulated expression upon MeJ application in macroblots as well as rt-Pcr analyses of B. juncea leaf-mrna. In arabidop-sis glucosinolates constitute the primary defense trait. In response to herbivory, MYr catalyzes cyanogenesis of glu-cosinolates to release toxic compounds such as nitriles, iso-thiocyanates, epithionitriles, and thiocyanates as a defense response in Brassicaceae (rask et al. 2000). Interestingly, chemotypes of glucosinolates in arabidopsis were found to be strongly correlated with geographical predominance of specialist aphids (Züst et al. 2012). Similarly, transcriptional activation of protease inhibitors including tPI as a primary defense response against herbivory has been documented in many plant taxa (Zavala et al. 2004). However, aphid infes-tation did not elicit any transcriptional activation of these defense genes even after 24 h of inoculation in B. juncea.

MeJ is known to activate jasmonate-mediated host defense and in B. juncea its application activated sev-eral candidate host defense genes. therefore, it was likely that the application of MeJ in susceptible B. juncea plants would confer quantitative resistance to aphids. B. juncea plants subjected to prior treatment with exogenous applica-tion of MeJ demonstrated strong antibiotic effects on the infesting aphids. the activated host defense, evident by significant transcriptional activation of jasmonate biosyn-thetic genes, redox genes, and the late defense genes within 1–5 h of MeJ treatment, reduced fecundity of the aphid population compared to the control. In tomato, applica-tion of MeJ significantly reduced green peach aphid popu-lations by inducing levels of defense-related proteins viz. leaf-POD and -PPO (Boughton et al. 2006). the second-ary metabolic enzymes POD, PPO, and Pal, which gen-erate phenolic compounds, mediate active defense against

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insect herbivores (Han et al. 2009). compared to untreated plants infested with aphids, in MeJ-treated B. juncea plants the levels of POD, PPO, and Pal activity remained high, which might be contributing to induced antibiosis. POD acts as a defense enzyme as well as an antioxidant. It is involved in the strengthening of the plant cell walls by lig-nifications and suberization to deter aphid feeding (Maf-fei et al. 2007). as an antioxidant enzyme, it catalyzes the oxidization of phenolic precursors to quinones by utilizing intracellular H2O2. Increased Pal and PPO activity implies elevated biosynthesis of phenylpropanoids and associated secondary metabolites related to antibiosis.

In conclusion, it is intriguing that the jasmonate-medi-ated host defense in B. juncea is not elicited in response to infestation by mustard aphid L. erysimi. But when elic-ited by the exogenous agent MeJ, it showed the potential to reduce population growth rates of L. erysimi. For future perspective, it will be interesting to address whether the effects on aphid populations are due to active suppression of host defense responses by L. erysimi or simply due to a lack of perception of aphid-associated molecular cues by the host plant. Furthermore, it seems likely that enhance-ment of the jasmonate signaling pathway and its respon-siveness to aphid-related cues could provide a general defense against aphids.

Acknowledgments this work was supported by national Fund for BSFara, Indian council of agricultural research; in-house research grant of the national research centre on Plant Biotechnology and a Junior research Fellowship to KM by Department of Biotechnol-ogy, Ministry of Science and technology, government of India. the authors acknowledge gregory Pearce for critically evaluating the manuscript.

References

abe H, Ohnishi J, narusaka M, Seo S, narusaka Y, tsuda S, Kob-ayashi M (2008) Function of jasmonate in response and tolerance of arabidopsis to thrip feeding. Plant cell Physiol 49:68–80

aebi H (1984) catalase in vitro. Method enzymol 105:121–126argandoña VH, chaman M, cardemil l, Muñoz O, Zúñiga ge, cor-

cuera lJ (2001) ethylene production and peroxidase activity in aphid-infested barley. J chem ecol 27:53–68

atri c, Kumar B, Kumar H, Kumar S, Sharma S, Banga SS (2012) Development and characterization of Brassica juncea–fruticulosa introgression lines exhibiting resistance to mustard aphid (Lipa-phis erysimi Kalt). BMc genet 13:104

Baldwin It (1998) Jasmonate-induced responses are costly but ben-efit plants under attack in native populations. Proc natl acad Sci USa 95:8113–8118

Beyer WF, Fridovich I (1987) assaying for superoxide dismutase activity: some large consequences of minor changes in condi-tions. anal Biochem 161:559–566

Bhadoria nS, Jakhmola SS, Dhamdhere SV (1995) relative suscepti-bility of mustard cultivars to Lipaphis erysimi in north west Mad-hya Pradesh (India). J entomol res 19:143–146

Bhattacharya r, Koramutla MK, negi M, Pearce g, ryan ca (2013) Hydroxyproline-rich glycopeptide signals in potato elicit

signalling associated with defense against insects and pathogens. Plant Sci 207:88–97

Boughton aJ, Hoover K, Felton gW (2006) Impact of chemical elicitor applications on greenhouse tomato plants and population growth of the green peach aphid, Myzus persicae. entomol exp appl 120:175–188

Boyko eV, Smith cM, thara VK, Bruno JM, Deng Y (2006) the molecular basis of plant gene expression during aphid invasion: wheat Pto- and Pti-like sequences are involved in interactions between wheat and russian wheat aphid (Homoptera: aphidi-dae). J econ entomol 99:1430–1445

Bradford MM (1976) a rapid and sensitive method for the quantifica-tion of microgram quantities of protein utilizing the principle of protein-dye binding. anal Biochem 72:248–254

carlsson J, lagercrantz U, Sundström J, teixeira r, Wellmer F, Meye-rowitz eM, glimelius K (2007) Microarray analysis reveals altered expression of a large number of nuclear genes in developing cyto-plasmic male sterile Brassica napus flowers. Plant J 49:452–462

casteel cl, Walling ll, Paine tD (2006) Behavior and biology of the tomato psyllid, Bactericerca cockerelli, in response to the Mi-1.2 gene. entomol exp appl 121:67–72

castillo FJ, Penel c, greppin H (1984) Peroxidase release induced by ozone in Sedum album leaves. Involvement of ca2+. Plant Phys-iol 74:846–851

christensen Sa, nemchenko a, Borrego e, Murray I, Sobhy IS, Bosak l, DeBlasio S, erb M, robert ca, Vaughn Ka, Herrfurth c, tumlinson J, Feussner I, Jackson D, turlings tc, engelberth J, nansen c, Meeley r, Kolomiets MV (2013) the maize lipoxy-genase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack. Plant J 74:59–73

creelman ra, Mullet Je (1997) Biosynthesis and action of jas-monates in plants. annu rev Plant Physiol Plant Mol Biol 48:355–381

Dean JD, goodwin PH, Hsiang t (2005) Induction of glutathione S- transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involve-ment of one in resistance. J exp Bot 56:1525–1533

Dickerson DP, Pascholati SF, Hagerman ae, Butler lg, nicholson rl (1984) Phenylalanine ammonia-lyase and hydroxyl cin-namate: coa ligase in maize mesocotyls inoculated with Hel-minthosporium maydis or Helminthosporium carbonum. Physiol Plant Pathol 25:111–123

Divol F, Vilaine F, thibivilliers S, amselem J, Palauqui Jc, Kusiank c, Dinant S (2005) Systemic response to aphid infestation by Myzus persicae in the phloem of Apium graveolens. Plant Mol Bio 57:517–540

Dogimont c, Bendahmane a, chovelon V, Boissot n (2010) Host plant resistance to aphids in cultivated crops: genetic and molec-ular bases, and interactions with aphid populations. c r Biol 333:566–573

ellis c, Karafyllidis I, turner Jg (2002) constitutive activation of jasmonate signaling in an arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae. Mol Plant Microbe Interact 15:1025–1030

Farmer ee, ryan ca (1992) Octadecanoid-derived signals in plants. trends cell Biol 2:236–241

götz S, garcía-gómez JM, terol J, Williams tD, nueda MJ, robles M, talón M, Dopazo M, conesa a (2008) High-throughput functional annotation and data mining with the Blast2gO suite. nucleic acids res 10:3420–3435

guerrieri e, Digilio Mc (2008) aphid-plant interactions: a review. J Plant Interact 3:223–232

Halitschke r, Baldwin It (2004) Jasmonates and related compounds in plant–insect interactions. J Plant growth regul 23:238–245

193Planta (2014) 240:177–194

1 3

Halkier Ba, gershenzon J (2006) Biology and biochemistry of glu-cosinolates. annu rev Plant Biol 57:303–333

Han Y, Wang Y, Bi Jl, Yang XQ, Huang Y, Zhao X, Hu Y, cai Qn (2009) constitutive and induced activities of defense-related enzymes in aphid-resistant and aphid-susceptible cultivars of wheat. J chem ecol 35:176–182

He J, chen F, chen S, lv g, Deng Y, Fang W, liu Z, guan Z, He c (2011) chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation. J Plant Physiol 168:687–693

Hogenhout Sa, ammar eD, Whitfield ae, redinbaugh Mg (2008) Insect vector interactions with persistently transmitted viruses. annu rev Phytopathol 46:327–359

Hori K, Wada a, Shibuta t (1997) changes in phenoloxidase activi-ties of the galls on leaves of Ulmus davidiana formed by Tetra-neura fusiformis (Homoptera: eriosomatidae). appl entomol Zool 32:365–371

Hossain Ma, Maiti MK, Basu a, Sen S, ghosh aK, Sen SK (2006) transgenic expression of onion leaf lectin gene in Indian mustard offers protection against aphid colonization. crop Sci 46:2022–2032

Kanrar S, Venkateswari J, Kirti PB, chopra Vl (2002) transgenic Indian mustard (Brassica juncea) with resistance to the mustard aphid (Lipaphis erysimi Kalt.). Plant cell rep 20:976–981

Karpinski S, Muhlenbock P (2007) genetic, molecular and physiolog-ical mechanisms controlling cell death, defenses, and antioxidant network in response to abiotic and biotic stresses in plants. comp Biochem Physiol a Mol Integr Physiol 146:60–66

Kempema la, cui X, Holzer FM, Walling ll (2007) arabidopsis transcriptome changes in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in responses to aphids. Plant Physiol 143:849–865

Klingler J, Powell g, thompson ga, Isaacs r (1998) Phloem specific aphid resistance in Cucumis melo line ar 5: effects on feeding behaviour and performance of Aphis gossypii. entomol exp appl 86:79–88

Klingler J, creasy r, gao l, nair rM, calix aS, Jacob HS, edwards Or, Singh KB (2005) aphid resistance in Medicago truncatula involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by nBS-lrr resistance gene analogs. Plant Physiol 137:1445–1455

Kusnierczyk a, Winge P, Jørstad tS, Ska Jt, rossiter Jt, Bones aM (2008) towards global understanding of plant defence against aphids—timing and dynamics of early Arabidopsis defence responses to cabbage aphid (Brevicoryne brassicae) attack. Plant cell environ 31:1097–1115

lagudah eS, Moullet O, appels r (1997) Map-based cloning of a gene sequence encoding a nucleotide binding domain and a leu-cine-rich region at the Cre3 nematode resistance locus of wheat. genome 40:659–665

lee SH, ahsan n, lee KW, Kim DH, lee Dg, Kwak SS, Kwon SY, Kim tH, lee BH (2007) Simultaneous overexpression of both cuZn superoxide dismutase and ascorbate peroxidase in trans-genic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. J Plant Physiol 164:1626–1638

lee Sc, lim MH, Kim Ja, lee SI, Kim JS, Jin M, Kwon SJ, Mun JH, Kim YK, Kim HU, Hur Y, Beom-Seok Park BS (2008) transcrip-tome analysis in Brassica rapa under the abiotic stresses using Brassica 24 K oligo microarray. Mol cells 26:595–605

leo FD, Bonade-Bottino Ma, ceci lr, gallerani r, Jouanin l (1998) Opposite effects on Spodoptera littoralis larvae of high expression level of a trypsin proteinase inhibitor in transgenic plants. Plant Physiol 118:997–1004

li c, Williams MM, loh Yt, lee gI, Howe ga (2002) resist-ance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiol 130:494–503

lukasik I, golawska S, Wojcicka a (2012) effect of cereal aphid infestation on ascorbate content and ascorbate peroxidase activity in triticale. Pol J environ Stud 21:1937–1941

Maffei Me, Mithöfer a, Boland W (2007) Insects feeding on plants: rapid signals and responses preceding the induction of phyto-chemical release. Phytochemistry 68:22–24

Marimuthu M, Smith cM (2012) Barley tolerance of russian wheat aphid (Hemiptera: aphididae) biotype 2 herbivory involves expression of defense response and developmental genes. Plant Signal Behav 7:382–391

Meihls ln, Handrick V, glauser g, Barbier H, Kaur H, Haribal MM, lipka ae, gershenzon J, Buckler eS, erb M, Köllner tg, Jander g (2013) natural variation in maize aphid resistance is associated with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity. Plant cell 25:2341–2355

Mhamdi a, Queval g, chaouch S, Vanderauwera S, Breusegem VF, noctor g (2010) catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. J exp Bot 61:4197–4220

Milligan SB, Bodeau J, Yaghoobi J, Kaloshian I, Zabel P, William-son VM (1998) the root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leu-cine-rich repeat family of plant genes. Plant cell 10:1307–1319

Moloi MJ, van der Westhuizen aJ (2006) the reactive oxygen spe-cies are involved in resistance responses of wheat to the russian wheat aphid. J Plant Physiol 163:1118–1125

Montesano M, Hyytiäinen H, Wettstein r, Palva et (2003) a novel potato defence-related alcohol:naDP+ oxidoreductase induced in response to Erwinia carotovora. Plant Mol Biol 52:177–189

nakano Y, asada K (1981) Hydrogen peroxide is scavenged by ascor-bate-specific peroxidase in spinach chloroplasts. Plant cell Phys-iol 22:867–880

nombela g, Williamson VM, Muniz M (2003) the root-knot nema-tode resistance gene Mi-1.2 of tomato is responsible for resist-ance against the whitefly Bemisia tabaci. Mol Plant Microbe Interact 16:645–649

Orozco-cardenas Ml, narvaez-Vasquez J, ryan ca (2001) Hydro-gen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, sys-temin, and methyl jasmonate. Plant cell 13:179–191

Park SJ, Huang Y, ayoubi P (2006) Identification of expression pro-files of sorghum genes in response to greenbug phloem-feed-ing using cDna subtraction and microarray analysis. Planta 223:932–947

Porta H, rocha-Sosa M (2002) Plant lipoxygenases. Physiological and molecular features. Plant Physiol 130:15–21

rask l, andréasson e, ekbom B, eriksson S, Pontoppidan B, Mei-jer J (2000) Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mol Biol 42:93–113

riemann M, Haga K, Shimizu t, Okada K, ando S, Mochizuki S, nishizawa Y, Yamanouchi U, nick P, Yano M, Minami e, takano M, Yamane H, Iino M (2013) Identification of rice allene oxide cyclase mutants and the function of jasmonate for defense against Magnaporthe oryzae. Plant J 74:226–238

rohwer cl, erwin Je (2010) Spider mites (Tetranychus urticae) per-form poorly on and disperse from plants exposed to methyl jas-monate. entomol exp appl 137:143–152

rossi M, goggin Fl, Milligan SB, Kaloshian I, Ullman De, William-son VM (1998) the nematode resistance gene Mi of tomato con-fers resistance against the potato aphid. Proc natl acad Sci USa 95:9750–9754

Seah S, Sivasithamparam K, Karakousis a, lagudah eS (1998) clon-ing and characterisation of a family of disease resistance gene analogs from wheat and barley. theor appl genet 97:937–945

Sekhon BS, ahman I (1993) Insect resistance with special reference to mustard aphid. In: lubana KS, Banga SS, Banga SK (eds)

194 Planta (2014) 240:177–194

1 3

Monographs on theoretical and applied genetics. Breeding oil-seed Brassicas, vol 19. Springer, new York, pp 206–221

Shekhawat K, rathore SS, Premi OP, Kandpal BK, chauhan JS (2012) advances in agronomic management of Indian mustard (Brassica juncea (l.) czernj. cosson): an overview. Int J agron. doi:10.1155/2012/408284

Shivaji r, camas a, ankala a, engelberth J, tumlinson JH, Williams WP, Wilkinson Jr, luthe DS (2010) Plants on constant alert: ele-vated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize. J chem ecol 36:179–191

Smith cM, Boyko eV (2007) the molecular bases of plant resistance and defense responses to aphid feeding: current status. entomol exp appl 122:1–16

Somssich Ie, Wernert P, Kiedrowski S, Hahlbrock K (1996) Arabi-dopsis thaliana defense-related protein elI3 is an aromatic alcohol:naDP+ oxidoreductase. Proc natl acad Sci USa 93:14199–14203

Stenzel I, Hause B, Maucher H, Pitzschke a, Miersch O, Ziegler J, ryan ca, Wasternack c (2003) allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato—amplification in wound signalling. Plant J 33:577–589

Stotz HU, Pittendrigh Br, Kroymann J, Weniger K, Fritsche J, Bauke a, Mitchell-Olds t (2000) Induced plant defense responses against chewing insects. ethylene signaling reduces resistance of

arabidopsis against egyptian cotton worm but not diamondback moth. Plant Physiol 124:1007–1017

taki n, Sasaki-Sekimoto Y, Obayashi t, Kikuta a, Kobayashi K, ainai t, Yagi K, Sakurai n, Suzuki H, Masuda t, takamiya K, Shibata D, Kobayashi Y, Ohta H (2005) 12-oxo-phytodienoic acid triggers expression of a distinct set of genes and plays a role in wound-induced gene expression in arabidopsis. Plant Physiol 139:1268–1283

tian D, Peiffer M, De Moraes cM, Felton gW (2014) roles of ethylene and jasmonic acid in systemic induced defense in tomato (Solanum lycopersicum) against Helicoverpa zea. Planta 239:577–589

Wasternack c (2007) Jasmonates: an update on biosynthesis, sig-nal transduction and action in plant stress response, growth and development. ann Bot 100:681–697

Zavala Ja, Patankar ag, gase K, Hui D, Baldwin It (2004) Manipu-lation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134:1181–1190

Züst t, Heichinger c, grossniklaus U, Harrington r, Kliebenstein DJ, turnbull la (2012) natural enemies drive geographic variation in plant defenses. Science 338:109–116