JXB Advance Access originally published online on June 1, 2007
Journal of Experimental Botany 2007 58(10):2537-2552; doi:10.1093/jxb/erm043
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Transcriptional responses of Arabidopsis thaliana ecotypes with different glucosinolate profiles after attack by polyphagous Myzus persicae and oligophagous Brevicoryne brassicae
nierczyk1
1Department of Biology, The Norwegian University of Science and Technology, Realfagbygget, 7491Trondheim, Norway
2Department of Biology, Imperial College at Wye, Wye, Kent TN25 5AH, UK
3School of Biological Sciences, University of Portsmouth, Portsmouth, Hants PO1 2DY, UK
* To whom correspondence should be addressed. E-mail: atle.bones{at}bio.ntnu.no
Received 22 January 2007; Accepted 7 February 2007
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Abstract |
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Plants are equipped with a range of defence mechanisms against herbivorous insects. In cruciferous species, jasmonic acid, salicylic acid, and ethylene along with glucosinolates and their hydrolysis products play important roles in plant protection and plant–insect communication. In turn, a number of herbivores have adapted to plants that contain glucosinolates. As a result of adaptation to their host plants, specialized insects may elicit different plant-inducible responses than generalists. Oligonucleotide microarrays and qRT–PCR analysis were used to characterize transcriptional profiles of Arabidopsis thaliana plants in response to infestation with a generalist aphid, Myzus persicae, or a cruciferous plant specialist, Brevicoryne brassicae. To find possible differences and similarities in molecular responses between plants differing in predominant glucosinolate hydrolysis products, three ecotypes of A. thaliana were chosen: Wassilewskija (Ws), Cape Verde Islands (Cvi), and Landsberg erecta (Ler), which, respectively, produce mainly isothiocyanates, epithionitriles, and nitriles. In all three ecotypes, general stress-responsive genes, genes belonging to octadecanoid and indole glucosinolate synthesis pathways were induced upon both generalist and specialist attack. By contrast, transcription of myrosinases, enzymes hydrolysing glucosinolates, was suppressed. The induction of the jasmonic acid synthesis pathway was strongest in Cvi, while the up-regulation of the indole glucosinolate synthesis pathway was highest in Ler, suggesting a slightly different defence strategy in these two ecotypes. Specialist and generalist infestations caused statistically significant differential regulation of 60 genes in Ws and 21 in Cvi. Among these were jasmonic acid and tryptophan synthesis pathway enzymes, and pathogenesis related protein (PR1). Insect no-choice experiments revealed lowered fitness of B. brassicae on Ler and Cvi in comparison to Ws, but no ecotype-dependent change in fecundity of M. persicae. Targeted studies employing constructs of GUS reporter gene under the control of promoters from CYP79B2 and CYP79B3 genes showed insect-specific induction of the indole glucosinolates synthesis pathway.
Key words: Aphid, generalist, indole glucosinolates, infestation, jasmonate, microarray, myrosinase, plant defence system, specialist
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Introduction |
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Phloem-feeding insects can cause severe damage to a great number of different agricultural crops. They can harm their hosts not only directly, by feeding on them, but also by transmitting viral diseases among individual plants. Aphids, which have a wide geographical distribution, are amongst the worst crop pests. They use anatomically adapted mouthparts, called stylets, for probing and exploring plant tissue as they search for sieve elements with nutritious sap (Pollard, 1973).
Plant genome responses to insect feeding are complex (Kessler and Baldwin, 2002). A review by Glazebrook (2001) characterizes at least three genetically distinguishable, but partially overlapping, inducible defence pathways: salicylic acid (SA)-dependent signalling, jasmonic acid (JA)/ethylene (ET)-dependent signalling, and resistance (R)-gene signalling. The SA- and JA/ET-dependent signalling pathways enable molecular cross-talk, but co-ordination between them and other stress responses is not fully understood. The octadecanoid pathway, a part of JA/ET signalling, is among those most affected by wounding and insect attack. A review by Halitschke and Baldwin (2004) summarizes regulation and function of JA signalling and its effect on herbivore performance. ET and abscisic acid (ABA) are believed to be positive regulatory factors in the octadecanoid pathway, while auxin is known to be a negative regulator of wound-induced JA-related responses (Walling, 2000), as it limits the duration of response to wounding (Rojo et al., 1998).
A distinctive defence system is present in cruciferous plants (Brassicaceae), which include many important crops and the model plant Arabidopsis thaliana. This system consists of nitrogen- and sulphur-containing secondary metabolites, called glucosinolates, and thioglucoside glucohydrolase enzymes (TGG), called myrosinases. Upon tissue damage, a range of toxic products is formed such as nitriles, isothiocyanates, thiocyanates, and epithionitriles (Bones and Rossiter, 1996, 2006; Wittstock and Halkier, 2002; Grubb and Abel, 2006; Halkier and Gershenzon, 2006). Because the glucosinolate-myrosinase system plays a major role in the defence strategy of cruciferous plants, the differences in composition of glucosinolate compounds are possibly of critical importance when assessing plant susceptibility to infestation. Various Arabidopsis ecotypes, originating from different geographical areas, have previously been shown to have diverse glucosinolate profiles (Kliebenstein et al., 2001; Lambrix et al., 2001) and different resistance to green peach aphid Myzus persicae (Poch et al., 1998). Studies of polymorphism of upstream regulatory elements in genes from different ecotypes suggest that they could be primary targets of natural selection, and contribute to regulation of genes involved in stress responses (Chen et al., 2005). However, studies comparing gene regulation in different ecotypes in response to insect feeding are lacking. Such a comparison should yield important new information about natural variation of Arabidopsis defence responses.
Several studies have been conducted to assess the specificity of Arabidopsis Col-0 genome responses to various abiotic and biotic stresses, including wounding, JA treatment, and infestation by pathogenic fungi, bacteria, or chewing and phloem-feeding insects (Reymond et al., 2000; Schenk et al., 2000; Moran and Thompson, 2001; Mikkelsen et al., 2003; Thilmony et al., 2006). A few reports explored comparative transcriptional profiling during exposure to generalist and specialist herbivores (Reymond et al., 2004; De Vos et al., 2005), but only two focused on plant–aphid interactions (Moran et al., 2002; Mewis et al., 2006). From these two, the second one describes changes in aliphatic glucosinolates biosynthesis. Moran and co-workers monitored the induction of a range of different Arabidopsis genes in response to feeding by the generalist pest Myzus persicae. Comparable studies employing the specialist Brevicoryne brassicae have, however, been restricted to a small number of genes analysed with macroarrays and do not provide a general comparison of plant reactions to generalist and specialist attacks. More extensive studies are therefore needed to acquire a complete picture of inducible responses of Arabidopsis challenged with M. persicae and B. brassicae. To create a comprehensive view of transcript changes during interactions with specialist and generalist pests, an experiment having two main objectives was designed.
The first objective was to compare global changes in gene-expression levels of three Arabidopsis thaliana ecotypes, Landsberg erecta (Ler), Wassilewskija (Ws), and Cape Verde Islands (Cvi) in response to aphid infestation. The ecotypes were chosen based on known differences in glucosinolate profiles (Kliebenstein et al., 2001; Lambrix et al., 2001). The expression patterns of three selected groups of defence-inducible genes were assessed: the first related to the octadecanoid pathway, the second being general stress responders, and the third being genes related to the myrosinase–glucosinolate system and the auxin synthesis pathway. These profiles were then examined to identify the ecotypes most and least responsive to infestation.
The second objective was to investigate the specificity of plant responces to a specialist pest of cruciferous species, the cabbage aphid (B. brassicae), in comparison to a generalist, the green peach aphid (M. persicae). By examining gene expression profiles in response to the specialist and generalist aphids, an attempt was made to determine if Arabidopsis is able to respond differently to these insects and thus adjust its defensive strategy according to the attacker.
To assess transcriptional profiles of infested versus control plants, custom-made oligonucleotide microarrays representing genes from 450 gene families were used, with a focus on defence pathways, signalling, and nutrient metabolism. Expression profiles of selected genes were confirmed by qRT–PCR. Insect-specific induction patterns of genes involved in indole glucosinolates (IG) synthesis pathway were assessed with the use of transgenic lines expressing CYP79B2:GUS and CYP79B3:GUS plants. In addition, in order to provide the biological context of the studied system, no-choice experiments were conducted with both aphid species to determine the suitability of the three ecotypes as hosts for M. persicae and B. brassicae.
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Materials and methods |
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Plant material
Three Arabidopsis thaliana ecotypes were used: Landsberg erecta (Ler), Cape Verde Islands (Cvi), and Wassilewskija (Ws). Single seed lines were derived from seeds produced by Lehle Seeds (Round Rock, USA; Ws: Catalogue No: WT-08A-09, Seed Lot No.: GH198-05; Ler: Catalogue No.: WT-04-14, Seed Lot No.: GH198-01; Cvi: Catalogue No. WT-18-1, Seed Lot No. GH192-22). Arabidopsis seeds were sown into 6 cm diameter pots filled with sterile soil mix (1.5 part soil, 1 part peat moss, and 0.5 part horticultural perlite, calcium 4 g l–1 of ready mixture). CYP79B2/CYP79B3:GUS seeds were sown as above or were sterilized according to standard procedures and plants were grown aseptically on agar medium containing MS basal salt mixture (Sigma), 3% (v/w) sucrose, and 0.7% (v/w) agar (pH 5.7). Plants were kept in growth rooms or growth chambers at 16/8 h (light/dark) photoperiod at 22/18 °C, 40/70% relative humidity, and 70/0 µmol m–2 s–1 light intensity.
Insects
Two species of aphids were used for infestation, the specialist aphid Brevicoryne brassicae or the generalist Myzus persicae. Aphid clones were maintained on Brassica napus plants in growth chambers at 16/8 h (light/dark) photoperiod at 22/18 °C, 40/70% relative humidity, and 70/0 µmol m–2 s–1 light intensity. One week before the experiment, aphids were transferred to Col-0 wild-type plants for adaptation to the new host species. For no-choice experiments aphids were reared on cabbage.
Infestation of different ecotypes
Plants that were at the beginning of the bolting stage, and that had developed between 10–12 rosette leaves (22–30-d-old, depending on the ecotype used), were used for all experiments. Four separate experiments (biological replicates) were performed for all ecotypes. Each biological replicate consisted of 28 individual plants. Insect bioassays were conducted in cages of transparent plexi-glass cylinders (20 cm diameter, 49 cm high) with a top of fine mesh gauze (mesh width: <0.2 mm), maintaining air exchange. Each cage contained seven pots with plants. Infested and control plants were maintained at the same conditions. In one experimental setup, 28 plants (four cages) were infested with B. brassicae, 28 plants with M. persicae, with the same number of plants maintained as the aphid-free controls. Fine mesh gauze with several 0.5 cm diameter holes distributed evenly on the surface, was fixed onto the inner walls of each cylinder above the plants. Caged plants were infested by transferring apterous aphids (adults and nymphs) to the gauze and allowing the insects to go through the holes and settle on the plants. After 1 d (or when each leaf was infested with approximately five aphids) the gauze was removed. The gauze was also used in the cages with control plants, but without aphids. Leaves were harvested 75 h after the start of the experiment (3 h to allow infestation, followed by 72 h of aphid feeding). Harvesting was always done between 15.00 h and 18.00 h. In aphid treatments, only leaves infested with approximately 8–12 aphids were harvested. The aphids were removed by washing the leaves with Milli-Q-filtered water. Harvested leaves were immediately frozen in liquid nitrogen and stored at –80 °C for RNA isolation.
RNA isolation, cDNA synthesis, and microarrays experiments
For each pair-wise combination, non-infested plants of the same ecotype were used as a control for plants infested with M. persicae and plants infested with B. brassicae. Relative gene expression values (expressed in log2 ratios) always represent the difference between the expression level of a given gene in infested plants and in the aphid-free control. RNA was isolated with Qiagen RNeasy Plant Midi Kit (Qiagen, Valencia, CA, USA) and quantified with Nanodrop ND 1000 (NanoDrop Technologies, Wilmington, DE, USA). The quality of RNA was assessed with the use of formaldehyde agarose gel electrophoresis. Total RNA (15 µg) and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) were used in a reverse transcription reaction. A 3DNA Array 350 kit (Genisphere Inc., Hatfield, PA, USA) was used for labelling. Microarray slides were printed by the Norwegian Microarray Consortium (Trondheim, Norway). They contained 2158 unique 70 mer oligos selected from the 3' end of Arabidopsis cDNAs. The high specificity oligonucleotide probes were designed with a Perl based program (Winge, unpublished). The oligonucleotides were produced by Operon (Alameda, CA, USA) or MWG (Ebersberg, Germany). The oligonucleotides were dissolved in MQ and 50% DMSO (40 pmol µl–1) and spotted in eight copies on amino silane coated UltraGaps slides (Corning, NY, USA) using a BioRobotics MicroGrid II robot (Genomic Solutions, MI, USA). All hybridizations were performed in a Tecan HS 4800 Hybridization Station (Tecan, Maennedorf, Switzerland) with the use of either 25% or 33% concentration of formamide in the final hybridization solution. The resulting images were analysed using GenePix 5.1 software (Axon Instruments, Union City, USA).
Statistical analysis
Gpr result files from the GenePix program were filtered and normalized prior to hypothesis testing. All spots flagged by GenePix as bad, not found, or absent were removed. A spot was also removed if the foreground signal was less than 1.5 times the background signal for one of the dyes. Several plots (including MA-plots, image plots of the log ratios, and the background signals) were made to assess the quality of the arrays. Print tip normalization (Yang et al., 2001) was applied to each array to correct for print tip and intensity-dependent effects. This procedure fitted a LOWESS curve (Cleveland et al., 1992; Cleveland and Loader, 1996) to scatter plots of the log ratio versus the average log intensity for each print tip sector and adjusted the log ratios according to the curves. Each oligonucleotide probe was printed eight times on each array, and the log ratios from these spots were combined so that one value was obtained for each gene and biological replicate. This was achieved by taking the median if three or more observations were available for an oligonucleotide probe. If only two or fewer were available, the value of the represented gene was declared as missing. The hypothesis testing was done with Limma (Smyth, 2005). One linear model was fitted for each ecotype. Each model had two parameters. One represented the difference between B. brassicae infested plants and untreated plants. The other measured the difference between M. persicae infested plants and untreated plants. The parameters and the contrast of B. brassicae infested plants versus M. persicae infested plants were tested for each gene. The resulting P-values were adjusted with Storey's q-value procedure (Storey, 2002). The false discovery rate (FDR) was controlled at 0.05 so that genes with q-value below 0.05 were declared as significant. Possible differences between plant responses to the two aphid species and among ecotypes, in terms of the numbers of up-regulated and down-regulated genes, were investigated using log-linear analysis (Sokal and Rohlf, 1981) implemented in SYSTAT (Wilkinson, 1988), with maximum-likelihood ratios employed for hypothesis testing. Four independent comparisons were made without correcting explicitly for multiple comparisons, and thus marginally significant differences should be interpreted with caution.
Real-time PCR analysis
Real-time PCR was used to evaluate the expression profiles of selected genes in at least two out of four biological replicates studied. SuperScript III reverse transcriptase (Invitrogen) and total RNA after DNAse treatment (Invitrogen) was used for cDNA synthesis. Gene-specific primers were designed to amplify a region of 119–227 bp (primer sequences are given in Supplementary Table S4 at JXB online). TIP41-like gene (At4g34270) (Czechowski et al., 2005) was used as a normalizer. Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA, USA) and iTaq SYBR Green Supermix With ROX (Bio-Rad) were used to run the three-step programme, which included (i) enzyme-activation at 95 °C for 3 min, (ii) 40 cycles of 95 °C for 30 s, 55 °C for 45 s, and (iii) 95 °C for 1 min, 55 °C for 45 s, 95 °C for 30 s for dissociation curve analysis. Each 25 µl reaction contained cDNA quantity corresponding to 0.0125 µg of RNA and 0.2 µM of each forward and reverse primer. Relative expression values were calculated using efficiency
Ct method.
Experiments with CYP79B2/CYP79B3:GUS plants, GUS staining and stylet tracks staining
Transgenic plants in Col-0 background, expressing GUS under control of the CYP79B2 or CYP79B3 promoters were kindly provided by John Celenza (Department of Biology, Boston University). For each construct two independent mutant lines were used for all experiments with similar results. Sterile cultures of 2-week-old plants were exposed for 72 h to MeJA treatment in 1.0 l glass beaker sealed with parafilm, by placing a sterile Eppendorf tube cup filled with 100 µl 95% MeJA solution (Aldrich Inc., Milwaukee, WI, USA) in the middle of an agar plate (a distance between all plants on the plate and MeJA source was equal). MeJA concentration was quantitatively determined in a gas phase by headspace SPME following GC-MS, based on a calibration curve of four MeJA concentrations to be 50 µmol l–1 volume. Control plants were kept in similar conditions, but water was used instead of MeJA. For wounding experiments or aphid infestations, 2-week-old plants moved from sterile cultures to medium containing 0.7% (v/w) agar (pH 5.7) or 4-week-old plants grown on soil were used. A fine, 52 µm in diameter, needle produced by pulling a glass pipette over a flame, was used for wounding. Whole plants were fixated in ice-cold acetone for 5 min, incubated in 0.1 M sodium phosphate, pH 7.0 and subsequently submerged for 18 h at 37 °C in a staining solution [(0.1 M sodium phosphate pH 7.0, 1% (v/w) Triton X-100, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM disodium EDTA pH 8.0, and 0.2 mM X-gluc (Duchefa, Haarlem, Netherlands)]. Chlorophyll was removed by subsequent washing with ethanol (70% v/w). Stylet tracks were stained with acid fuchsin as described by Brennan and co-workers (Brennan et al., 2001) with the exception that no sectioning was done. Images were captured with SPOT CCD camera (Diagnostic Instruments, Sterling Heights, MI) coupled to microscope (Nikon S800) or with Nikon COOLPIX camera coupled to Nikon (C-DSD230) stereo system.
Aphid no-choice experiments
Twenty-day-old plants of each of the ecotypes were used for this experiment. One first instar nymph was placed on a rosette leaf of each plant. Plants with aphids were maintained in plexi-glass cylinders in the same conditions as in infestation experiments, with an exception that each plant was additionally contained in a seed collector to prevent movement of aphids from one plant to another. After 11 d the number of progeny on each plant was counted. The data represent results from two independent experiments. The no-choice experiments assessing ecotype effects on fecundity of the two aphid species were analysed using 1-way and 2-way (factorial) analyses of variance (ANOVA) and post hoc tests using procedures implemented in SYSTAT (Wilkinson, 1988).
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Quantitative comparative analysis of microarray results
Aphid infestations of all three ecotypes resulted in significant changes in the plants' transcriptomes. To compare quantitative changes across all ecotype–aphid combinations, all genes that had statistically significant changes in expression levels were divided into two groups: up-regulated and down-regulated, and subsequently into two subgroups: those where the change in expression level was larger than or less than 2-fold (Fig. 1A). A log-linear analysis was performed on these four groups separately. Aside from the total number of genes up-regulated and genes repressed more than 2-fold in the Ler ecotype, the number of responding genes was always slightly, but not statistically significantly, higher after infestation with M. persicae than B. brassicae (non-significant main aphid effect: log-likelihood X2=0.94–3.65, P=0.82–0.30, df=3). Considering the number of regulated genes, there was no statistical evidence for ecotype-specific differences in their responses to the two aphid species (non-significant ecotype-by-aphid interaction terms: log-likelihood X2=0.16–1.79, P=0.92–0.41, df=2). The comparison of the three ecotypes showed, however, a very strong main ecotype effect (significant ecotype terms: log-likelihood X2=19.6–135.9, P
0.001, df=4). The largest number of all reacting transcripts was found in Ws, with the lowest found in Ler. But if the quantities of transcripts with at least 2-fold induction levels were compared, Cvi turned out to be the most responsive of the ecotypes, with the numbers of up-regulated genes 25% and 57% larger in comparison to Ws and Ler attacked by M. persicae and 22% and 51% larger in these ecotypes attacked by B. brassicae, respectively. The quantitative comparison of genes commonly up-regulated or down-regulated between the ecotypes revealed highest agreement between responses of Ws and Cvi and lowest between Cvi and Ler (Fig. 1B; see Supplementary Fig. S1 at JXB online).
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Quantitative real-time PCR analysis
Seven statistically differentially regulated genes were selected for further analysis with a quantitative real-time PCR method. The same RNA pools from at least two biological replicates were used for microarrays and for qRT-PCR analysis. The results show good correlation with gene expression profiles obtained from microarray data (Fig. 2).
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Induction of genes in JA-dependent wound-signal transduction pathway
Several genes from the octadecanoid pathway leading to the production of JA were up-regulated upon infestation both with M. persicae and B. brassicae (Fig. 3). Two key enzymes were strongly induced, particularly in Cvi; these were allene oxide synthase (AOS, Cyp 74A) (At5g42650) and lipoxygenases (LOX) (At1g72520, At1g17420), which are involved in the release of fatty acids from complex membrane lipids (Leon et al., 2001) and which commit them to the JA synthesis pathway or the generation of 6-carbon volatiles (Gatehouse, 2002). By contrast, induction of lipoxygenase 2 (LOX2) (At3g45140) was highest in Ler. The transcript of 12-oxophytodienoate reductase (OPR1) (At1g76680) showed the strongest response in Cvi. OPR1 was shown to be less efficient than OPR3 in converting 12-oxo-phytodienoic acid (OPDA) (Schaller et al., 2000) and therefore OPR3 is believed to play the main role in the JA biosynthesis pathway. The crystal structure revealed, however, that OPR1 has substrate specificity similar to OPR3 (Breithaupt et al., 2001) and thus its role in JA synthesis or oxylipin signaling cannot be ruled out. It was also found that transcripts of phospholipases D gamma (PLD gamma1: At4g11850, PLD gamma2: At4g11830, PLD gamma3: At4g11840) had accumulated in infested plants. PLDs have an important role in the release of linolenic acid from membrane lipids (Walling, 2000) and may mediate wound induction of JA (Wang et al., 2002). Co-regulation of these functionally related genes possibly indicates elevated production of JA. In addition, up-regulation of octadecanoids-inducible coronatine-regulated chlorophyllase (CORI1) (At1g19670) and coronatine-regulated tyrosine aminotransferase (TAT, CORI3) (At4g23600) also confirms the activation of the octadecanoid pathway upon aphid feeding. The latter enzyme is involved in the synthesis of tocopherols, which are radical scavengers in plants and have previously been reported to be induced by methyl jasmonate (MeJA) and methyl-12-oxo-phytodienoic acid (MeOPDA) (Lopukhina et al., 2001; Sandorf and Hollander-Czytko, 2002).
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Pathogenesis- and oxidative stress-related genes
One of the major responders in the infestation experiments of Ler and Ws ecotypes was SA-dependent pathogenesis-related protein 1 (PR-1) (At2g14610) (Laird et al., 2004). Surprisingly, this gene was down-regulated in Cvi infested with M. persicae (Fig. 4). The PR4 gene (At3g04720) that codes for a hevein-like protein (HEL) was up-regulated in Ws and Ler. Its expression is stimulated by the accumulation of cytotoxins at the site of lesion formation in host–pathogen interactions (Almeras et al., 2003). The induction of the gene related to regulatory protein for PR gene (NPR-1 related) (At5g45110) was higher for Cvi than for Ws and Ler. In addition, a number of calmodulins (CM), a putative calmodulin (PCM) (At3g25600), and calmodulin-related protein (CMR) (At1g66400) were more induced in Cvi. Most of calmodulin-like (CML), but not calmodulin-binding (CMB) proteins were generally up-regulated (for details see Supplementary Table S1 at JXB online).
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The expression of a gene from the ethylene biosynthesis pathway encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS6) (At4g11280) was increased at similar levels in response to both aphids, and was most pronounced in the Cvi ecotype. In addition, the ethylene-responsive element-binding protein (ERF1) (At4g17500) was up-regulated in all ecotypes. Induction of this protein integrates signals from the ethylene- and jasmonate-signalling pathways (Lorenzo et al., 2003) resulting in the transcriptional activation of defence-related genes.
Nitrate reductases (NR1, At1g77760; NR2, At1g37130) and respiratory burst oxidase protein D (RBOHD) (At5g47910) were strongly induced in the infested Cvi ecotype, and the heat shock factor 21 protein (HSF21) (At4g18880) was also more induced in Cvi (Table 1). The expression of a group of 13 glutathione-S-tranferase genes (GST and GSTU) and putative GST genes was changed upon infestation. These stress- and wound-responsive genes are important in the process of detoxification of many compounds and reactive oxygen species (Walling, 2000). Most of glutathione-associated genes were up-regulated, especially GSTU7 and GSTF6, but interestingly two genes: GSTU 18 (At1g10360) and GSTU 20 (At1g78370) were down-regulated in all ecotypes (Fig. 4). The expression of glutathione-conjugate transporter (MRP4) (At2g47800) was significantly increased for Ws and Cvi.
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Expression profiles of glucosinolate- and auxin-synthesis pathway-related genes
Infestation with both the generalist and specialist aphid resulted in a significant induction of several genes involved in the synthesis of tryptophan-derived IG (Fig. 5). The tryptophan synthase
subunit (TSA1) (At3g54640) was up-regulated in all of the experiments, with the exception of Cvi infested with M. persicae. Anthranilate synthase β subunit (put. ASB) (At1g25083), another enzyme involved in tryptophane synthesis, was also induced, mainly in Ws and Ler. The expression of two cytochrome P450 enzymes CYP79B2 (At4g39950) and CYP79B3 (At2g22330), catalysing the conversion of tryptophan to indole-3-acetaldoxime (IAOx) (Hull et al., 2000; Mikkelsen et al., 2000), was substantially increased. Interestingly, the induction of CYP79B2 was highest in Ler, while CYP79B3, although generally less induced, showed highest expression in Ws and Cvi. CYP83B1 (At4g31500), which has a crucial role in the production of IG (Hansen et al., 2001; Bak et al., 2001) was also up-regulated. By contrast, genes involved in the production of aliphatic glucosinolates: CYP83A1 (At4g13770) and CYP79F1 (At1g16410) were generally not up-regulated in response to aphid infestation (Table 1). The expression of the UDP-glucose: thiohydroximate S-glucosyltransferase (UGT74B1) (At1g24100), which is required for the production of desulphoglucosinolates, was just slightly increased in Ler. Desulphoglucosinolates are thought to be myrosinase-resistant phloem transport forms of glucosinolates (Brudenell et al., 1999; Grubb et al., 2004).
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As CYP83B1 is the metabolic branch point in IG and auxin biosythesis in Arabidopsis, and overexpression of CYP83B1 leads to auxin deficit (Bak et al., 2001), the induction of CYP83B1 possibly decreases the production of indole-3-acetic acid (IAA). It was found that the auxin-responsive protein, IAA3 (At1g04240) was strongly down-regulated in our experiment, especially for Ws and Ler (Fig. 5). Also the expression of IAA17 (At1g04250) was decreased in Ws and Ler (Table 1). These results indicate that the production of IAA in infested plants can be lowered compared to control plants. The expression level of some IAA hydrolases, especially IAR3 (At1g51760), ILL6 (At1g44350), and ILL3 (At5g54140) was elevated (Fig. 5). These enzymes are capable of hydrolysing amide-linked conjugates of IAA, which are putative storage or inactivated forms of auxin (Davies et al., 1999; LeClere et al., 2002; Rampey et al., 2004). Accumulation of transcripts of IAA hydrolases indicates a response to the reduced level of IAA.
Induction patterns of CYP79B2 and CYP79B3 genes are aphid specific
As previous work reported the increased level of IG in response to MeJA treatment (Kliebenstein et al., 2002), the changes observed here in gene expression level of key enzymes from the IG pathway may have been explained as a consequence of an increase in JA synthesis. To investigate if this was the case in our experiment, the induction pattern of CYP79B2 and CYP79B3 genes in response to MeJA and insect attack was compared, using transgenic plants expressing GUS under the control of either the CYP79B2 or the CYP79B3 promoter (Ljung et al., 2005). Exposure to MeJA resulted in a strong, restricted to vascular tissue, induction of both CYP79B2 and CYP79B3 (Fig. 6A–C, E–G). Aphid feeding caused a dramatic change in GUS expression in CYP79B2:GUS plants and a very weak change in CYP79B3:GUS plants. The expression patterns of CYP79B2 after insect attack were clearly distinct from those mediated by MeJA, and, moreover, slightly different for the two aphid species. CYP79B2 induction by B. brassicae was visible mainly around the probing sites (Fig. 6L, P), while M. persicae appeared to cause more broad activation, involving a bigger leaf area (Fig. 6K, N, O). The hardly observable induction of CYP79B3 was comparable in the case of both aphids and was restricted to the vascular tissue of younger leaves. MeJA treatment gave a much stronger activation of CYP79B3 promoter than insect feeding. Wounding by piercing did not induce expression, neither of CYP79B2 nor CYP79B3 (Fig. 6J, M).
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Myrosinases and myrosinase-related proteins
The genes coding for the two myrosinases expressed in the green parts of Arabidopsis, TGG1 (At5g26000) and TGG2 (At5g25980), were down-regulated in all experiments (except for TGG2 in Cvi in both infestations and Ler attacked by B. brassicae, where the regulation was not statistically significant) (Table 1). Generally, M. persicae altered expression levels of genes coding for myrosinases and myrosinase-related proteins more than B. brassicae. The epithiospecifier modifier1 (ESM1) (At3g14210) (Zhang et al., 2006) was also down-regulated in all ecotypes. By contrast, myrosinase binding protein 1 (MBP1) (At1g52040) and a putative myrosinase-binding protein belonging to a jacalin lectin family (put. MBP) (At1g52000) (see Supplementary Table S1 at JXB online) were induced after infestation in Ws and Cvi. The protein encoded by At1g52000 is closely related to a vegetatively expressed and wound-/JA-inducible MBP from Brassica napus (CAA70587 [GenBank] ). The epithiospecifier protein (ESP) (At1g54040), catalysing the formation of epithionitriles during glucosinolate hydrolysis (Zabala et al., 2005), was down-regulated in almost all experiments, while a gene encoding for a kelch repeat protein related to ESP (ESP-like) (At3g07720) (Table 1), for which the function has not been characterized, was slightly up-regulated, particularly in Cvi and Ler.
Genes differentially regulated in plants attacked by B. brassicae versus plants attacked by M. persicae
Sixty genes were differentially regulated (q-value 0.05 and the difference in log2 ratio at least 0.5) by the feeding of B. brassicae and M. persicae in the Ws ecotype and 21 in Cvi ecotype. A list of all the genes is available in the supplementary material (see Supplementary Tables S2 and S3 at JXB online). In Table 1 differentially regulated genes are marked with a star. No transcripts met the required threshold for the q-value in the case of Ler. It is, however, possible that an increased number of biological replicates would help to reveal the differences in response to aphids in the Ler ecotype as well (Jørstad et al., 2007). In the Cvi ecotype, two genes from the octadecanoid pathway, AOS and OPR1, were more induced upon the generalist aphid attack. By contrast, PR-1 was down-regulated by M. persicae but up-regulated by B. brassicae. Among the genes that were differentially expressed in the Ws ecotype were TGG1 and ESM1, both of which were more repressed as a result of feeding by the generalist. On the contrary, CORI3 was markedly up-regulated to a greater degree by the B. brassicae attack. Interestingly, of all genes that were statistically differentially regulated by M. persicae and B. brassicae, just four transcripts were common to the Ws and Cvi ecotypes; these were (At4g35750) encoding a sec14 domain protein, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) (ALDH11A3) (At2g24270), a putative copper amine oxidase (At1g31690), and adenosylhomocysteinase 2 (SAH hydrolase 2) (At3g23810).
Aphid fitness in no-choice experiments
Asexual fecundity has been adopted as a measure of fitness of the two aphid species on the different Arabidopsis ecotypes. M. persicae reproduced similarly on all three ecotypes (1-way ANOVA: P=0.31), while the number of B. brassicae progeny was slightly reduced on Ler and more then twice reduced in Cvi in comparison to Ws (1-way ANOVA with post hoc contrasts: Ws versus Ler P=0.034; Ws versus Cvi: P=0.001; Ler versus Cvi P=0.002) (Fig. 7). When following the Bonferroni procedure only the difference in fecundity of B. brassicae on Cvi in comparison to Ws and Ler were found to be significant at the 0.05 level. These results suggest that some factors influencing performance of B. brassicae do not have the same effect on fitness of M. persicae.
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During their life cycle, plants are often exposed to attack by insect herbivores. To be able to survive and reproduce, plants are equipped with various defence mechanisms. A crucial part of this system consists of inducible responses, which are triggered by pest attack. The complexity of the reaction to insect feeding makes it difficult to study the specificity of defensive mechanisms. Changes in gene expression profiles can be a consequence of the response of genes directly and indirectly involved in insect resistance, but will also include general stress-responders and genes not believed to be involved in stress or defence responses (Haile et al., 1999; Hui et al., 2003). In this paper, our analysis was focused on transcriptional changes of genes involved in well-characterized defence signalling pathways. A 72 h aphid-infestation period was chosen based on previous reports from similar experiments, which have revealed strong induction of many genes at this time point (Moran and Thompson, 2001; Moran et al., 2002). It is possible, however, that the expression profile of several genes (especially those with a very short induction time) would be different, when measured at an earlier or later stage of infestation. A time-course experiment would be needed to get a more complete picture of the transcriptional responses to the aphid infestation.
The induction of JA-synthesis pathway is strongest in the Cvi ecotype
Our results confirm the important role of the octadecanoid pathway in response to aphid feeding in all three ecotypes. The accepted mechanism for activation of this pathway implicates the damage of cell membranes as a starting point for the release of linolenic acid from membrane lipids. Linolenic acid then acts as a precursor to the synthesis of JA (Schaller, 2001). However, aphids, in contrast to chewing insects, have evolved a feeding strategy that minimizes cell damage. During probing, the aphid stylet proceeds mostly intercellularly, leaving the cell membrane intact. Our studies show that, despite this sophisticated feeding strategy, aphids are not able to prevent a cascade of plant defences, which has been shown to be triggered by wounding. In addition to the stylet piercing some cells along its path, which allows the aphid to examine their contents, other factors may induce plant defence responses. In particular, chemical stimuli caused by the injection of watery saliva into a targeted cell or the deposition of the salivary sheath along the stylet's way may elicit plant responses. Recent studies have shown that M. persicae is a potent inducer of transcriptional responses when compared with various microbial pathogens and herbivorous insects, although the aphid generates weaker symptoms (De Vos et al., 2005). To determine whether the degree of induction of the JA synthesis pathway upon infestation differs between the ecotypes studied, the expression level of the enzymes LOX, AOS, OPR1, and CORI3 was compared. Our results revealed that activation of the octadecanoid pathway is strongest in Cvi (Fig. 3). Strong activation of JA-related defence mechanisms correlated with a lowered fecundity of B. brassicae feeding on Cvi compared with the two other ecotypes. However, the same effect was not observed in the case of M. persicae, which has been shown previously to be less sensitive to JA-mediated plant defences (Mewis et al., 2005).
Pathogenesis- and oxidative stress-related genes are affected in all ecotypes
Previous reports have indicated that Col-0 plants infested with M. persicae showed strong induction of a group of PR and SA-dependent genes and moderate responses from genes connected to the JA synthesis pathway (Moran and Thompson, 2001; Moran et al., 2002). The increase in expression level of the PR-1 gene in Ws and Ler in our experiment is consistent with studies done by Moran and co-workers. In infestations of these ecotypes with both M. persicae and B. brassicae, the induction level of PR-1 is stronger than of any gene from the JA pathway. Promoter–reporter gene studies with the use of transgenic Arabidopsis lines expressing PR-1:GUS constructs have confirmed strong induction of PR-1 around the feeding sites of M. persicae in Col-0 plants (De Vos et al., 2005). In contrast, in Cvi B. brassicae caused a weaker induction of PR-1 compared to most of the enzymes from the JA synthesis pathway, and interestingly, M. persicae caused its down-regulation. Studies of cereal crops attacked by aphids revealed stronger induction of PR proteins in resistant plant genotypes (van der Westhuizen et al., 1998; Forslund et al., 2000). This finding is consistent with the fact that some of PR proteins, due to their special properties, may directly harm plant pests. PR4 protein, which was up-regulated in our experiment in Ws and Ler, contains hevein domain and may bind to chitin present in exoskeleton of insects (Asensio et al., 2000).
Nitrate reductases (NR1 and NR2), which were highly up-regulated mostly in Cvi, have been recently confirmed to have an important function in response to environmental stimuli. Bright and co-workers (2006) showed that nitrate reductase is the major source of nitric oxide (NO) in ABA-mediated H2O2 synthesis. Both NO and H2O2 are key molecules involved in different signal transduction pathways coupled to wound and pathogen defence processes and might influence inducible responses (Wasternack et al., 2006). Co-regulation of nitrate reductases and respiratory burst oxidase protein D (RBOPD), which is required for accumulation of reactive oxygen intermediates in plant defense responses (Torres et al., 2002), can reflect recently described partnership between NO and H2O2 during the induction of programmed cell death (Zago et al., 2006). The process of H2O2 accumulation during aphid feeding can be possibly triggered by injection of polygalacturonases present in aphid saliva (Miles, 1999). These enzymes can digest polysaccharides of pierced cell walls inducing cascade of H2O2 accumulation, which in turn can lead to an up-regulation of GSTs involved in detoxification of reactive oxygen species.
A number of general stress-responsive genes were induced more strongly in the Cvi ecotype (Fig. 4), possibly contributing to higher resistance of this ecotype to B. brassicae attack.
IG synthesis pathway is up-regulated but myrosinases are down-regulated
Glucosinolate breakdown products have been shown to suppress microbial growth (Brabban and Edwards, 1995; Tierens et al., 2001) and affect insect feeding (Barth and Jander, 2006). These products often act as repellents to generalist pests, but they can also be important host cues for specialist herbivores (Miles et al., 2005). Infestation both with M. persicae and B. brassicae resulted in a significant increase in the expression of genes from the IG synthesis pathway, but a moderate decrease in transcripts of glucosinolate hydrolysing myrosinases. Up-regulation of the tryptophane biosynthetic genes, such as anthranilate synthase (AS) and tryptophan synthase subunit (TSA1) in response to feeding by M. persicae is supported by other reports in the literature (Moran and Thompson, 2001; Moran et al., 2002). The level of IG has also been shown to increase in response to wounding and insect feeding in other Brassicaceae species (Koritsas et al., 1991; Hopkins et al., 1998; Bartlet et al., 1999).
The elevated level of glucosinolates is often linked to JA synthesis. Brader and co-workers showed that the induction of IG in response to Erwinia carotovora is jasmonate-dependent (Brader et al., 2001). Induction of enzymes from the glucosinolate synthesis pathway and the level of IG have been reported to increase in response to MeJA or JA treatments both in Brassica napus (Doughty et al., 1995) and Arabidopsis (Mikkelsen et al., 2003; Mewis et al., 2005). The increased production of JA would therefore be expected to promote the production of glucosinolates. Indeed, our experiments with plants expressing either CYP79B2:GUS or CYP79B3:GUS showed a strong responsiveness of both genes to MeJA exposure. In addition to possible jasmonate-mediated up-regulation, localized induction of CYP79B2 during aphid attack near feeding places, may be jasmonate-independent. In a recent report jasmonate-independent accumulation of aliphatic glucosinolates was observed after aphid feeding (Mewis et al., 2006).
Our experiments revealed the strongest activation of IG pathway in the Ler ecotype. Interestingly, studies of MeJA-induced IG production in different Arabidopsis ecotypes have shown that Ler was more responsive than the other ecotypes (Kliebenstein et al., 2002). Higher inductiveness could explain why the IG pathway is up-regulated to a higher degree in Ler compared with the two other ecotypes, despite not having the highest induction of the JA synthesis pathway. The same or even lower level of JA would simply lead to greater up-regulation of the IG pathway in Ler than in other ecotypes.
Notably, the two genes coding for the Arabidopsis myrosinases, TGG1 and TGG2, proved to have redundant function (Barth and Jander, 2006), were in our experiment moderately down-regulated. This observation is consistent with studies focusing on the regulation of myrosinase transcripts in Brassica napus plants infested with B. brassicae (Pontoppidan et al., 2003). These authors observed a lowered expression of a gene coding for myrosinase after infestation with B. brassicae. Whether the observed down-regulation of these genes affects glucosinolate content is, however, unknown. The MyAP–epithiospecifier modifier1 (ESM1) (At3g14210) was also down-regulated upon infestation with M. persicae and B. brassicae. ESM1 was recently shown to be a quantitative trait locus, which directs glucosinolates hydrolysis toward isothiocyanate production (Zhang et al., 2006). Interestingly, ESM1, ESP, TGG1, and TGG2 (all proved to be functionally associated with the glucosinolate–myrosinase system) have a similar expression profile. One possible explanation for the decrease in their expression level could be that aphids, along with salivary secretions, introduce some inhibitors directly or indirectly capable of changing the expression level of selected genes. The differential regulation of TGGs and ESP, as compared to MBP1 and the ESP-related protein (At3g07720) is also noteworthy. The functional role of the ESP-related protein has not yet been determined and it might well be involved in processes other than glucosinolate degradation.
Transcriptional reprogramming upon generalist and specialist pest attack—is it real?
The plant defence system is thought to respond to insect herbivory in a specific manner. It has been demonstrated that the various experimental techniques used by researchers to artificially mimic tissue damage caused by herbivory are generally not sufficient to induce strong insect-specific reactions (Reymond et al., 2000; Pontoppidan et al., 2005). One example is the CYP79B2 gene not being locally inducible by piercing with a sterile needle, but reacting to piercing with aphids stylets, accompanied by other factors connected to aphid feeding. This suggests that additional factors such as salivary secretions or specific aphid/insect recognition systems in plants are needed to influence the expression of specific genes. Moreover, the induction of CYP79B2 gives a slightly different pattern in response to specialist and generalist attacks. This distinction is likely a consequence of well-described differences in the feeding behaviour of these two species (Cole, 1997) or possible differences in the composition of salivary secretions. It is not known, however, if the IG or their hydrolysis products are toxic to the two species of aphids. The answer to this question is of importance, but lies beyond the scope of this study.
It is also possible that B. brassicae uses glucosinolates as a nutrition source, since this specialist is adapted to the plants containing glucosinolates. The myrosinase that was discovered in B. brassicae (Jones et al., 2001) has activity towards plant-derived glucosinolates in in vitro enzymatic assays (Francis et al., 2002; Husebye et al., 2005) and this may be the case in vivo as well. The elevated level of IG around the feeding place would make it more attractive to other members of the colony. This model corresponds well to feeding behaviour of B. brassicae. All insects in the colony are very compactly grouped around their feeding places, and new-born nymphs usually stay close to parents.
A quantitative comparison of expression profiles of other genes responding to generalist and specialist aphids revealed a moderately stronger response to M. persicae. Most of the genes from the main inducible pathways were induced by the two aphids, suggesting that similar defence mechanisms were triggered. When comparing the general and JA-related stress responses induced by aphids in the three ecotypes, Cvi turns out to be the most responsive. Its suitability as a host plant for the specialist aphid is also lower compared to Ws and Ler. B. brassicae have specialized to feed on a narrow range of cruciferous plants. The co-evolution with its plant hosts has resulted in the acquisition of adaptive mechanisms, probably making the aphid more resistant to glucosinolate-myrosinase defence system. Specialization may be costly from an evolutionary point of view and the price paid may be a slightly reduced ability to deal with general (JA-related) plant defensive mechanisms. M. persicae, the generalist lacking host specialization, is on the other hand reproducing equally well on the three ecotypes. Studies monitoring aphids populations growth on JA-signalling impaired mutant (coi-1) and wt plants confirmed that M. persicae was more resistant to JA-related plant defence than B. brassicae (Mewis et al., 2005). The important role of the octadecanoid pathway has recently been implicated also in the resistance of Medicago truncatula against a legume specialist aphid, Acyrthosiphon kondoi (Gao et al., 2007). The increased activation of the IG pathway and possibly elevated levels of indole glucosinolates in Ler appear also not to affect M. persicae fitness. These results, together with studies of Barth and Jander (2006), who did not find any effect of knocking out the Arabidopsis foliar myrosinases on M. persicae and B. brassicae performance, suggest that aphids are able to prevent, avoid, and/or cope with toxic glucosinolate breakdown products.
Interestingly, the attack by the generalist influenced to a greater degree the expression of the main enzymes from octadecanoid pathway in Cvi and myrosinase and ESM1 in Ws and it caused opposite expression of PR-1 gene in Cvi. The reason for this remains unexplored and deserves further experimentation.
Finally, it is important to stress, that the generalist and the specialist herbivores in our experiments were each represented by only one species of aphid. It is possible that the detected differences in plant response can be due to aphid-species differences and might not be applicable to other generalist and specialist insects. Further research preferably on both a genomic and a metabolomic level, is needed to determine whether plants are able to adjust their defence strategy according to the specialization of the attacker.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsSupplementary materialReferences |
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It has been demonstrated that three Arabidopsis thaliana ecotypes, which differed in their predominant glucosinolate hydrolysis products, activated similar groups of defensive genes upon aphid attack. However, both quantitative and qualitative differences in transcriptome changes were found between ecotypes. Although Ws showed the greatest number of affected genes, Cvi showed greater changes in expression profiles of affected genes and was the least suitable host for the oligophagous aphid. Plant response profiles also appeared to be fine-tuned to the attacker. The polyphagous aphid M. persicae generally caused slightly more changes in gene expression levels than did the oligophagous B. brassicae. This finding, together with aphid specific regulation of CYP79B2 and CYP79B3 genes, indicates that different attackers induce distinguishable different changes in the Arabidopsis transcriptome.
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Supplementary material |
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The following supplementary material is available for this article at JXB online:
Fig. S1. Venn diagrams showing the number of common genes up-regulated or down-regulated between the three different Arabidopsis ecotypes in response to M. persicae and B. brassicae attack.
Table S1. Combined list of expression values for 1197 genes having q-values below 0.05 at least for one ecotype–aphid combination. Values declared as missing or having q-values over 0.05 are marked as NA.
Table S2. List of genes differentially regulated by M. persicae and B. brassicae in the Ws ecotype, presenting gene expression values in response to both attackers.
Table S3. List of genes differentially-regulated by M. persicae and B. brassicae in Cvi ecotype, presenting gene expression values in response to both attackers.
Table S4. Primers used for quantitative real-time PCR analysis.
This material is available as part of the online article on http://jxb.oxfordjournals.org.
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Acknowledgements |
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The authors would like to thank John Celenza for providing CYP79B2/CYP79B3:GUS seeds, Torfinn Sparstad for the help with microarray experiments, and Jens Rohloff for measuring MeJA concentration. Special thanks to Ralph Kissen and Nancy Bazilchuk for useful comments on the manuscript, and Wac
aw Kunierczyk for help with editing of figures. This work was supported by the Biotechnology and Functional genomics (FUGE) programmes of the Norwegian Research Council through grants NFR 159959, 164583 and 151991. |
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