Development of a spruce cDNA microarray
Based on the generation of spruce ESTs, we have developed the first spruce cDNA microarray composed of 9720 cDNA elements selected from five cDNA libraries representing shoot tips, xylem and roots of various developmental stages, and bark and phloem treated with MeJA and mechanical wounding (Table 1). Treatment with mechanical wounding and MeJA can simulate, at least in part, insect attack in spruce (Martin et al. 2002, 2003; Miller et al. 2005). Clones on the array were selected from a sequence assembly of c. 12 000 3′-end ESTs (CAP3 software, Huang & Madan 1999) and represent c. 5500 unique genes. Functional annotation of array elements has been assigned according to The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) Arabidopsis peptide set [BLASTX expect value (E) < 1e−05]. Overall, only 5570 (57.4%) of 9720 spotted cDNAs have similarity to Arabidopsis known or predicted protein sequences, which could be a result of the long evolutionary distance between gymnosperm and angiosperm genomes, the expression of unique genes in conifers that are not found in the Arabidopsis genome, and/or the bias in the spruce EST dataset towards 3′ non-coding regions. Similarity of spruce transcripts with previously annotated angiosperm genes, or the lack thereof, is the topic of a separate paper (S. Ralph & J. Bohlmann, unpublished data). Low similarity of many conifer genes with Arabidopsis was also previously found for loblolly pine (Kirst et al. 2003) and presents a challenge to interpreting the biological functions of differentially regulated transcripts in a conifer system.
Table 1. cDNA libraries used for constructing the spruce 9.7K cDNA microarray
|cDNA library||Tissue/Developmental stage||Species (Genotype)||No. on 9.7K array|
|WS-ES-A-1||Young shoots harvested from 25-year-old treesa||Picea glauca (PG-29)||3577|
|WS-PS-A-2||Flushing buds, young shoots and mature shoots harvested from 25-year-old treesa||P. glauca (PG-29)||1232|
|WS-X-A-3||Early (15 June), mid (10 July) and late (17 August) season outer xylem harvested from 25-year old treesa||P. glauca (PG-29)||2336|
|IS-B-A-4||Bark tissue (with phloem and cambium) harvested after razor blade wounding and treatment with 0.01% methyl jasmonate. Tissue was collected 0 (untreated), 3, 6 and 12 h post-treatmentb.||P. glauca × engelmannii (Fal-1028)||1290|
|SS-R-A-5||Young growth (terminal 1–3 cm) and mature growth (distal to terminal 1–3 cm) rootsb||Picea sitchensis (Gb2–229)||1269|
Microarray transcriptome profiling of Sitka spruce responses to insect herbivory and wounding
We have previously established that feeding by white pine weevils on Sitka spruce induces selected genes in the oleoresin terpenoid defence pathway, specifically TPS genes, a few genes in the octadecanoid signalling pathway and a family of DIR proteins (Miller et al. 2005; Ralph et al. 2006b). To extend this analysis with global transcriptome profiling, we used the spruce 9.7K cDNA microarray to examine changes in gene expression in spruce in response to insect herbivory and mechanical wounding. We also extended our previous analysis with the inclusion of two insect species, larvae of the spruce budworm and adult white pine weevils, which have different feeding patterns and consume different spruce tissues. Spruce budworms feed on the very young, green shoot tips and foliage, while weevils are stem-boring insects that drill into the bark of apical leaders to feed on phloem tissues. Clonal Sitka spruce saplings were subjected to either mechanical wounding of bark by razor blade, feeding on bark by stem-boring weevils or feeding on green leaders by defoliating budworms, with insects caged on trees using mesh bags (Fig. 1). In this initial transcriptome analysis, we selected the time point of 2 d (48–52 h) after the onset of insect feeding, based on earlier observations of strong induction of TPS and DIR genes at this time point (Miller et al. 2005; Ralph et al. 2006b). For comparison, and to assess the overall dynamics of an insect-induced transcriptome response in spruce, we also measured gene expression at an early time point (3 h) after the onset of budworm feeding. In contrast to insects, which required at least 1 d of continuous feeding to cause substantial tissue damage in our experiments, mechanical wounding causes rapid tissue damage. We therefore used a one-day time point for the analysis of wound-induced responses.
Figure 1. Herbivory experiment set-up under greenhouse conditions. Insects were caged under mesh bags placed on Sitka spruce saplings (a). Weevils inflict damage by boring into bark of the stem and feeding on phloem (b), whereas budworms sever and consume young needles and green shoot tips (c). Scale bars indicate approximate size.
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Differentially expressed (DE) genes were selected using two criteria: fold-change between treatment and control > 1.5x and P-value < 0.05. To estimate the false discovery rate (FDR), we calculated q-values (Storey & Tibshirani 2003) and found the FDR for budworm feeding at 3 and 52 h, weevil feeding at 48 h and mechanical wounding at 24 h to be 19.0, 3.1, 5.1 and 2.4% at P = 0.05, diminishing to 10.7, 0.8, 0.7 and 0.3% at P = 0.001, respectively (Supplementary Table S2). For a complete list of expression data for all genes represented on the microarray see Supplementary Table S2. It should be noted that although microarrays measure transcript abundance, this could be the result of changes in the transcription rate or of changes in the rate of transcript degradation. For brevity, these will collectively be referred to as changes in gene expression. The annotation of cDNA elements on the array as shown in Supplementary Table S2 is derived from annotation of each individual EST sequence, whereas annotation for genes described in Table 2 and Figs 4–6 is derived from a CAP3 sequence assembly of c. 80 000 3′-end spruce ESTs. The latter approach was chosen to improve annotation to Arabidopsis by using longer, continguous spruce sequences. Figure 2 illustrates the overall transcriptional response in shoot tips 3 and 52 h after the onset of budworm feeding, as well as in bark tissue 24 h after mechanical wounding or 48 h after the onset of weevil feeding. After only 3 h of budworm feeding, 358 elements were DE (3.6% of the transcriptome analysed; 221 up-regulated, 137 down-regulated) compared to 3490 (35.9% of the transcriptome analysed; 2040 up-regulated, 1450 down-regulated) after 52 h, with 244 elements commonly DE at both time points. In comparison, 2382 (24.5% of the transcriptome analysed; 1305 up-regulated, 1077 down-regulated) and 3089 (31.7% of the transcriptome analysed; 1682 up-regulated, 1407 down-regulated) elements were DE in response to weevil feeding and mechanical wounding, respectively. These results demonstrate substantial overall changes of the host plant transcriptome at 2 d after onset of feeding for each of the two insect–spruce host interactions.
Table 2. Selected herbivore- or wounding-responsive array elements
|Clone ID||BLASTX versus Arabidopsis||AGI code||E-value||Fig. 3b class||Mechanical at 24 h||Weevil at 48 h||Fig. 3a class||Budworm at 3 h||Budworm at 52 h|
|No significant match to Arabidopsis|
|IS0011_A15||No significant match||n.a.||n.a.||•||94.83|| 0.001||0.003||14.55||< 0.001||0.003||•||2.50|| 0.009||0.143||13.45|| 0.006||0.011|
|WS0044_F23||No significant match||n.a.||n.a.||•|| 0.53|| 0.020||0.013|| 0.77|| 0.064||0.061||•||0.30|| 0.026||0.163|| 0.17|| 0.001||0.008|
|WS0034_E23||No significant match||n.a.||n.a.||•|| 0.29|| 0.006||0.006|| 0.30|| 0.006||0.015||•||0.91|| 0.159||0.286|| 6.52|| 0.019||0.018|
|WS0044_G09||No significant match||n.a.||n.a.||•|| 7.09|| 0.006||0.006|| 1.77|| 0.023||0.032||•||1.38|| 0.002||0.116|| 0.01||< 0.001||0.002|
|Biological process unknown|
|WS0014_J09||Photoassimilate-responsive protein||At5g52390||e-10||•|| 1.10|| 0.019||0.012|| 0.80|| 0.083||0.072||•||0.80|| 0.005||0.126||10.26|| 0.012||0.014|
|WS0018_A07||Plantacyanin||At2g02850||e-25||•|| 0.31||< 0.001||0.002|| 0.28||< 0.001||0.003||•||0.82|| 0.053||0.193|| 1.37|| 0.052||0.031|
|WS0038_N22||Auxin-regulated protein||At2g33830||e-14||•|| 0.34||< 0.001||0.003|| 0.40||< 0.001||0.003||•||0.53|| 0.020||0.155|| 0.78|| 0.149||0.064|
|WS0017_F02||Low temperature and salt responsive||At3g05890||e-13||•|| 0.28|| 0.011||0.009|| 0.30|| 0.001||0.008||•||0.96|| 0.503||0.467|| 1.50|| 0.083||0.043|
|IS0014_J19||Lateral organ boundaries domain protein||At1g07900||e-20||•|| 0.89|| 0.109||0.043|| 8.43|| 0.002||0.009||•||1.55|| 0.074||0.213|| 4.18|| 0.041||0.027|
|WS0015_F13||Phosphate-responsive protein||At5g64260||e-84||•|| 2.81|| 0.001||0.003|| 2.79||< 0.001||0.005||•||2.44|| 0.009||0.143|| 7.58|| 0.001||0.008|
|WS0016_C06||GDSL-motif lipase||At5g33370||e-77||•|| 1.08|| 0.634||0.175|| 1.11|| 0.416||0.226||•||0.99|| 0.993||0.610||18.56|| 0.010||0.014|
|WS0039_N07||Gibberellin-regulated protein||At2g39540||e-16||•|| 0.82|| 0.063||0.029|| 0.57|| 0.090||0.076||•||1.78|| 0.055||0.195||13.37|| 0.013||0.015|
|WS0012_O19||Arabinogalactan||At5g53250||e-05||•|| 0.68|| 0.045||0.022|| 4.43|| 0.007||0.017||•||1.68|| 0.031||0.170|| 8.45|| 0.011||0.014|
|IS0012_L22||Haloacid dehalogenase hydrolase||At5g59480||e-28||•|| 3.00|| 0.032||0.018|| 2.40||< 0.001||0.006||•||2.19|| 0.001||0.107|| 8.01|| 0.003||0.010|
|WS00111_P23||Patatin||At2g26560||e-62||•|| 0.10|| 0.007||0.007|| 0.82|| 0.229||0.148||•||0.97|| 0.816||0.567|| 1.18|| 0.498||0.157|
|WS0038_G12||Xyloglucan endo-1,4-β-D- glucanase||At4g25810||e-71||•|| 0.28|| 0.010||0.008|| 2.65||< 0.001||0.004||•||2.10|| 0.001||0.116||18.50|| 0.017||0.017|
|WS00110_E02||GAP3DH||At1g42970||e-23||•|| 0.48||< 0.001||0.003|| 0.47|| 0.003||0.012||•||0.89|| 0.155||0.282|| 0.19|| 0.007||0.012|
|WS0018_O18||Fructose-1,6-biphosphate aldolase||At4g38970||e-99||•|| 0.62|| 0.003||0.005|| 0.66|| 0.002||0.011||•||0.92|| 0.041||0.178|| 0.28||< 0.001||0.002|
|WS0013_N23||Galactinol synthase||At2g47180||e-81||•|| 0.99|| 0.856||0.221|| 0.63|| 0.015||0.025||•||1.05|| 0.289||0.369|| 0.25|| 0.002||0.009|
|WS0019_N05||Fructose-1,6-biphosphatase||At1g43670||e-100||•|| 0.42|| 0.021||0.013|| 0.51|| 0.015||0.124||•||0.92|| 0.171||0.295|| 0.23|| 0.002||0.008|
|WS0041_E18||Transketolase||At3g60750||e-127||•|| 1.37|| 0.016||0.011|| 1.19|| 0.210||0.139||•||1.03|| 0.740||0.545|| 0.27||< 0.001||0.002|
|WS0015_L24||Hydroxypyruvate reductase||At1g68010||e-55||•|| 0.81|| 0.021||0.013|| 0.94|| 0.121||0.094||•||0.99|| 0.971||0.605|| 0.28||< 0.001||0.008|
|WS0013_D08||Iron-sulphur complex protein||At4g22220||e-61||•|| 0.65|| 0.010||0.008|| 1.03|| 0.807||0.354||•||0.91|| 0.137||0.267||14.82|| 0.001||0.008|
|WS0039_G18||Cellulose synthase||At1g55850||e-51||•|| 1.62|| 0.018||0.012|| 0.88|| 0.540||0.271||•||0.83|| 0.092||0.230|| 0.17|| 0.003||0.009|
|WS0015_E01||Glutamine synthase||At5g37600||e-143||•|| 1.28|| 0.207||0.072|| 0.74|| 0.072||0.066||•||0.92|| 0.069||0.209|| 0.18|| 0.004||0.010|
|WS0019_O08||Homocysteine S- methyltransferase||At5g17920||e-13||•|| 4.87||< 0.001||0.002|| 2.53|| 0.004||0.014||•||1.11|| 0.014||0.151|| 3.81||< 0.001||0.008|
|WS00113_A19||VLCFA condensing enzyme||At1g68530||e-74||•|| 0.94|| 0.439||0.131|| 0.72|| 0.147||0.108||•||0.88|| 0.043||0.179||13.85|| 0.001||0.008|
|WS0016_L07||Calcium-binding EF hand||At3g10300||e-52||•|| 0.98|| 0.927||0.235|| 0.90|| 0.620||0.299||•||0.97|| 0.610||0.507||13.97|| 0.006||0.011|
|WS00110_A15||Carbonic anhydrase||At1g08080||e-39||•||15.83|| 0.001||0.003||15.30||< 0.001||0.006||•||1.27|| 0.005||0.128|| 9.41|| 0.001||0.008|
|IS0013_C18||Expansin||At1g69530||e-51||•|| 5.90|| 0.002||0.004|| 0.66|| 0.024||0.032||•||0.84|| 0.342||0.398|| 2.75|| 0.012||0.014|
|WS0015_F04||β-galactosidase||At3g13750||e-55||•|| 5.26||< 0.001||0.002|| 0.92|| 0.746||0.337||•||1.12|| 0.370||0.410|| 0.64|| 0.038||0.026|
|WS00113_D16||α-galactosidase||At5g08370||e-95||•|| 8.80|| 0.001||0.003|| 2.52|| 0.043||0.046||•||1.07|| 0.352||0.403|| 7.27|| 0.001||0.008|
|WS00113_F20||Enoyl-CoA hydratase||At1g06550||e-43||•|| 4.30|| 0.002||0.004|| 3.34||< 0.001||0.004||•||0.90|| 0.095||0.234|| 5.16|| 0.003||0.009|
|IS0011_B10||Acid phosphatase||At4g29270||e-54||•||24.76||< 0.001||0.003||11.45||< 0.001||0.004||•||3.59|| 0.019||0.153||25.88|| 0.002||0.009|
|WS00111_J06||Photosystem II||At1g44575||e-49||•|| 0.63|| 0.002||0.004|| 0.39|| 0.002||0.010||•||0.86|| 0.003||0.126|| 0.35|| 0.010||0.013|
|WS00113_A22||Photosystem I subunit VI||At1g52230||e-35||•|| 0.52|| 0.048||0.023|| 0.53|| 0.017||0.026||•||1.03|| 0.787||0.559|| 0.35|| 0.009||0.013|
|WS0022_L16||Chlorophyll a-b binding protein||At1g29930||e-94||•|| 0.74|| 0.006||0.006|| 0.45||< 0.001||0.004||•||0.86|| 0.483||0.461|| 0.60|| 0.095||0.047|
|WS0024_C24||Light-regulated protein||At3g26740||e-16||•|| 0.43||< 0.001||0.002|| 0.34||< 0.001||0.004||•||0.73||< 0.001||0.107|| 0.24||< 0.001||0.006|
|WS0024_M05||Ferredoxina||At1g60950||e-41||•|| 0.47|| 0.007||0.007|| 0.38||< 0.001||0.004||•||0.90|| 0.447||0.444|| 0.41|| 0.004||0.010|
|WS0042_C02||Oligopeptide transporter||At1g22540||e-35||•||12.13|| 0.001||0.003||12.60||< 0.001||0.002||•||1.38|| 0.082||0.220|| 2.02|| 0.100||0.049|
|IS0011_I18||ABC proteina||At3g28360||e-32||•|| 5.07|| 0.003||0.005|| 9.68||< 0.001||0.004||•||2.18|| 0.049||0.189|| 4.97|| 0.001||0.008|
|WS0018_I09||Lipid transfer protein||At1g62500||e-25||•|| 4.71||< 0.001||0.002|| 3.75||< 0.001||0.005||•||1.36|| 0.005||0.126|| 6.61|| 0.014||0.015|
|WS0015_C05||Lipid transfer protein||At5g48485||e-07||•|| 0.25||< 0.001||0.002|| 0.46|| 0.013||0.023||•||0.87|| 0.109||0.247|| 0.16|| 0.004||0.010|
|WS0013_M24||Lipid transfer protein||At5g59310||e-23||•|| 0.88|| 0.287||0.093|| 1.12|| 0.502||0.258||•||0.85|| 0.133||0.264||34.02|| 0.003||0.009|
|WS00111_M12||AP2 transcription factora||At3g20310||e-09||•|| 5.23|| 0.011||0.009|| 2.83|| 0.013||0.023||•||1.26|| 0.056||0.196|| 3.09|| 0.014||0.015|
|IS0013_F06||Basic helix-loop-helix transcription factor||At5g51780||e-05||•||11.89|| 0.004||0.005|| 9.78|| 0.009||0.019||•||0.98|| 0.497||0.465|| 1.75|| 0.002||0.008|
|WS0042_F06||MADS-box transcription factor||At2g45660||e-14||•|| 0.79|| 0.193||0.068|| 0.97|| 0.873||0.372||•||1.03|| 0.409||0.429|| 5.57|| 0.013||0.015|
|WS0044_J05||bZIP transcription factora||At1g75390||e-20||•||10.15|| 0.001||0.003|| 6.36|| 0.003||0.012||•||1.12|| 0.058||0.197|| 1.07|| 0.729||0.210|
|Response to stress|
|IS0012_N03||Lectin||At1g52030||e-12||•||13.14||< 0.001||0.002|| 1.04|| 0.459||0.243||•||1.11|| 0.577||0.494|| 0.45|| 0.043||0.028|
|WS0021_I16||β-1,3-glucanase||At2g05790||e-114||•|| 1.12|| 0.134||0.051|| 1.07|| 0.184||0.127||•||0.95|| 0.361||0.408||11.44|| 0.002||0.009|
|WS0017_B07||β-1,3-glucanase||At2g01630||e-41||•|| 4.99|| 0.001||0.004|| 4.73|| 0.002||0.010||•||1.10|| 0.222||0.328|| 1.85|| 0.078||0.041|
|IS0013_A20||Late embryogenesis abundant protein||At4g02380||e-05||•||13.94|| 0.002||0.004||24.98||< 0.001||0.075||•||4.24||< 0.001||0.126||20.02||< 0.001||0.007|
|IS0013_J03||Class IV chitinasea||At3g54420||e-57||•||10.42|| 0.004||0.005||13.91||< 0.001||0.004||•||0.68|| 0.069||0.209|| 4.92|| 0.010||0.013|
|IS0013_G04||Thaumatin||At1g19320||e-29||•|| 7.75|| 0.004||0.005|| 4.58||< 0.001||0.002||•||0.72|| 0.129||0.261|| 0.11|| 0.002||0.009|
|WS0019_E03||Protease inhibitor||At5g43580||e-08||•|| 3.18|| 0.003||0.005|| 2.34||< 0.001||0.004||•||1.02|| 0.668||0.526|| 0.56|| 0.054||0.032|
|IS0012_P21||Class II heat shock protein||At5g12020||e-30||•|| 0.17||< 0.001||0.003|| 0.37||< 0.001||0.003||•||0.76|| 0.061||0.201|| 0.92|| 0.286||0.104|
|WS0023_A18||β-glucosidase||At3g18080||0||•|| 1.21|| 0.037||0.020|| 1.91|| 0.005||0.014||•||1.25|| 0.026||0.163|| 6.25|| 0.009||0.013|
|Detoxification, redox processes|
|WS0017_M23||lactoylglutathione lyase (glyoxalase I)||At1g80160||e-44||•||10.44|| 0.001||0.003|| 7.67||< 0.001||0.003||•||1.64|| 0.003||0.126||18.48|| 0.002||0.009|
|WS00110_M24||Peroxidase||At5g05340||e-69||•|| 3.67|| 0.002||0.004|| 1.78|| 0.012||0.022||•||1.15|| 0.164||0.289|| 6.81|| 0.014||0.015|
|WS0019_N09||Peroxidase||At1g71695||e-38||•|| 7.94|| 0.001||0.003|| 2.95|| 0.006||0.016||•||1.33|| 0.098||0.237|| 0.96|| 0.702||0.203|
|WS0037_N15||Methylenetetrahydrofolate reductase||At3g59970||e-71||•|| 4.59||< 0.001||0.002|| 2.33|| 0.002||0.010||•||1.12|| 0.001||0.116|| 4.73||< 0.001||0.007|
|WS0034_I08||Thioredoxin||At3g51030||e-27||•|| 1.90|| 0.005||0.006|| 1.64|| 0.001||0.008||•||1.12|| 0.268||0.357|| 6.78|| 0.005||0.011|
|WS0016_L21||Thioredoxin||At3g02730||e-14||•|| 0.55|| 0.037||0.019|| 0.42||< 0.001||0.003||•||0.82|| 0.060||0.200|| 0.26||< 0.001||0.007|
|WS0018_G23||Superoxide dismutase||At1g08830||e-64||•|| 0.91|| 0.060||0.028|| 0.89|| 0.093||0.078||•||1.04|| 0.142||0.271|| 0.25||< 0.001||0.008|
|WS0023_I24||Glycolate oxidase||At3g14420||e-66||•|| 1.66|| 0.004||0.005|| 0.82|| 0.014||0.023||•||1.04|| 0.486||0.462|| 0.28|| 0.001||0.008|
|WS0021_L21||Cytochrome b5||At5g25080||e-31||•|| 2.44|| 0.001||0.003|| 2.41||< 0.001||0.004||•||0.99|| 0.991||0.609|| 6.99|| 0.003||0.009|
|IS0014_M04||Glutathione S-transferase||At1g10360||e-26||•|| 3.08|| 0.004||0.005|| 4.12|| 0.002||0.009||•||0.99|| 0.965||0.604|| 0.77|| 0.186||0.075|
|WS0041_G15||Oxidoreductase||At4g23340||e-28||•||11.89||< 0.001||0.002|| 7.04|| 0.008||0.018||•||1.04|| 0.376||0.414|| 1.41|| 0.231||0.088|
|IS0011_J03||Oxidoreductase||At1g23740||e-41||•|| 5.26||< 0.001||0.002|| 1.15|| 0.431||0.232||•||0.29|| 0.001||0.107|| 0.38||< 0.001||0.008|
|WS0015_K19||Cytochrome P450||At1g33730||e-22||•|| 1.44|| 0.006||0.006|| 7.23|| 0.018||0.027||•||1.04|| 0.676||0.529|| 1.81|| 0.165||0.069|
|IS0012_L15||Cytochrome P450||At4g22690||e-28||•||34.14|| 0.002||0.004||33.07|| 0.001||0.007||•||1.48|| 0.114||0.251||23.13|| 0.017||0.017|
|Octadecanoid and ethylene signalling|
|IS0014_J20||Lipoxygenasea||At1g72520||e-111||•|| 4.24|| 0.005||0.006|| 4.47|| 0.009||0.019||•||1.72|| 0.062||0.202|| 5.61|| 0.005||0.011|
|WS0023_N05||Allene oxide synthase||At5g42650||e-52||•|| 4.12||< 0.001||0.002|| 2.62|| 0.002||0.010||•||1.18|| 0.168||0.292|| 1.87|| 0.036||0.025|
|WS0039_A09||Allene oxide cyclasea||At3g25770||e-62||•|| 4.36||< 0.001||0.003|| 8.30||< 0.001||0.002||•||5.11|| 0.002||0.123||18.78|| 0.002||0.009|
|WS0035_O19||S-adenosylmethionine synthase||At4g01850||e-64||•|| 7.16|| 0.002|| 0.004|| 2.77|| 0.001||0.009||•||1.14|| 0.092||0.230|| 5.42||< 0.001||0.008|
|WS00112_P16||ACC oxidase||At1g77330||e-50||•|| 6.57|| 0.001|| 0.003|| 7.95|| 0.002||0.009||•||1.24|| 0.042||0.178|| 3.50||< 0.001||0.008|
|WS00112_G07||Shikimate kinase||At2g21940||e-54||•|| 3.17|| 0.014||0.010|| 2.53|| 0.002||0.009||•||1.34||0.013||0.149|| 5.69|| 0.006||0.011|
|WS0038_H08||DAHP synthasea||At1g22410||e-106||•|| 2.61|| 0.005||0.006|| 3.31|| 0.001||0.008||•||1.37||0.013||0.151||14.16|| 0.001||0.008|
|IS0011_E16||Phenylalanine ammonia lyasea||At3g53260||e-43||•||43.05|| 0.001||0.003||12.56||< 0.001||0.004||•||1.78||0.057||0.197|| 5.97|| 0.002||0.008|
|WS0031_M22||Cinnamate-4-hydroxylasea||At2g30490||e-46||•||12.89||< 0.001||0.002|| 6.43||< 0.001||0.004||•||1.43||0.028||0.164||15.49|| 0.001||0.008|
|WS0031_C20||4-coumarate-CoA ligase||At3g21240||e-78||•|| 3.84|| 0.002||0.004|| 3.27||< 0.001||0.006||•||0.90||0.332||0.393|| 6.04|| 0.001||0.008|
|WS0041_M02||Caffeoyl-CoA O-methyltransferase||At4g34050||e-73||•|| 3.18|| 0.001||0.003|| 2.24|| 0.001||0.008||•||1.06||0.416||0.431|| 6.44|| 0.003||0.010|
|IS0014_O19||Caffeoyl-CoA O-methyltransferase||At4g34050||e-43||•|| 0.35||< 0.001||0.003|| 0.38||< 0.001||0.006||•||0.68||0.028||0.164|| 1.94|| 0.009||0.013|
|WS0021_F13||Caffeic acid O-methyltransferase||At1g51990||e-50||•||53.42||< 0.001||0.002||19.10||< 0.001||0.003||•||0.98||0.866||0.576|| 0.82|| 0.410||0.136|
|IS0011_E08||Laccase/diphenol oxidasea||At2g30210||e-50||•||63.86||< 0.001||0.002||21.02||< 0.001||0.002||•||2.77||0.109||0.248||19.09||< 0.001||0.006|
|WS0016_N04||Laccase/diphenol oxidasea||At5g05390||e-64||•||55.20||< 0.001||0.002||15.61||< 0.001||0.005||•||3.28||0.066||0.206||24.87|| 0.001||0.008|
|WS0031_H14||Dirigenta||At1g65870||e-45||•|| 0.79|| 0.014||0.010|| 0.65|| 0.021||0.030||•||0.80||0.037||0.175|| 4.71|| 0.023||0.020|
|IS0013_K10||Dirigenta||At1g64160||e-48||•||68.19||< 0.001||0.002||12.72||< 0.001||0.003||•||1.33||0.021||0.155|| 3.28|| 0.065||0.036|
|IS0013_L11||Chalcone synthasea||At5g13930||e-174||•|| 1.99|| 0.005||0.006|| 2.71|| 0.002||0.010||•||1.07||0.441||0.442|| 2.28|| 0.017||0.017|
|WS0011_B22||Chalcone isomerase||At5g05270||e-55||•|| 2.67||< 0.001||0.002|| 3.67||< 0.001||0.005||•||1.13||0.274||0.361|| 3.59|| 0.001||0.008|
|WS00110_P10||Anthocyanin 5-aromatic acyltransferase||At5g23940||e-39||•|| 3.15|| 0.010||0.008|| 2.42|| 0.001||0.007||•||0.90||0.197||0.312|| 3.37|| 0.001||0.008|
|WS0022_G09||Flavonoid 3-hydroxylase||At5g07990||e-45||•|| 1.74||< 0.001||0.002|| 2.21|| 0.004||0.013||•||0.92||0.106||0.245|| 4.69|| 0.001||0.008|
|WS0014_M03||Leucoanthocyanidin dioxygenase||At4g22880||e-88||•|| 1.46|| 0.006||0.006|| 1.69|| 0.003||0.012||•||0.91||0.089||0.226||12.72||< 0.001||0.006|
|WS0016_K11||Dihydroflavonol 4-reductasea||At5g42800||e-67||•|| 3.35|| 0.001||0.003|| 2.46||< 0.001||0.004||•||1.14||0.332||0.393|| 2.62||< 0.001||0.007|
|WS00111_A06||Geranylgeranyl diphosphate synthasea||At4g36810||e-57||•|| 3.45||< 0.001||0.003|| 1.62|| 0.004||0.014||•||1.12||0.312||0.384||13.07|| 0.001||0.008|
|WS00111_K11||Geranylgeranyl diphosphate synthase||At4g36810||e-56||•|| 0.45|| 0.004||0.005|| 0.46||< 0.001||0.006||•||0.80||0.001||0.116|| 0.66|| 0.120||0.055|
|WS0017_E16||Terpene synthasea||At3g14490||e-06||•||16.76||< 0.001||0.002|| 4.75||< 0.001||0.003||•||3.13||0.021||0.155|| 8.83|| 0.004||0.010|
|WS00111_H11||Terpene synthase||At1g48800||e-07||•||12.69||< 0.001||0.002|| 2.54||< 0.001||0.004||•||1.88||0.006||0.134|| 1.66|| 0.031||0.023|
|A complete list of array elements is given in Supplementary Table S2.|
|Colour scale from dark green to dark red correlates with fold-change expression.|
|aExpression determined by real-time PCR (Fig. 7 and Table 3).|
|ABC, ATP-Binding Cassette; AGI, Arabidopsis Gene Index; FC, fold-change; P, P-value; Q, q-value; GAP3DH, glyceraldehyde-3-phosphate dehydrogenase; VLCFA, very long chain fatty acid; bZIP, basic-leucine zipper; ACC, 1-aminocyclopropane-1-carboxylate; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate.|
Figure 4. Metabolic pathway scheme for octadecanoid biosynthesis. Arrows represent enzymatic reactions and boxes represent metabolic products. Each row of four coloured boxes adjacent to arrows represents an expressed sequence tag (EST) on the spruce 9.7K array, with individual boxes corresponding to the relative fold-change between a treatment and control for each type of treatment from left to right: mechanical wounding of bark 24 h, weevil feeding on bark 48 h, budworm feeding of green shoot tips 3 h, budworm feeding of green shoot tips 52 h. The colour scale indicates fold-change differences in gene expression between treated and control samples. Contig (c) numbers derived from a CAP3 sequence assembly of c. 80 000 3′-end spruce ESTs are provided adjacent to colour bars, along with expect values that represent BLASTX scores to The Arabidopsis Information Resource (TAIR) Arabidopsis thaliana peptide set. The ‘+’ or ‘–’ signs within boxes indicate statistical significance (P-value < 0.05) of up- or down-regulation, respectively. ESTs marked with an asterisk were also examined by real-time PCR (Fig. 7 and Table 3). A complete list of spruce ESTs with ID numbers listed in the same order (i.e. grouped by enzyme, top to bottom) as they appear in the figure is provided in Supplementary Table S3.
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Figure 5. Metabolic pathway scheme for terpenoid biosynthesis. Arrows represent enzymatic reactions and boxes represent metabolic products. Each row of four coloured boxes adjacent to arrows represents an expressed sequence tag (EST) on the spruce 9.7K array, with individual boxes corresponding to the relative fold-change between a treatment and control for each type of treatment from left to right: mechanical wounding of bark 24 h, weevil feeding on bark 48 h, budworm feeding of green shoot tips 3 h, budworm feeding of green shoot tips 52 h. The colour scale indicates fold-change differences in gene expression between treated and control samples. Contig (c) numbers derived from a CAP3 sequence assembly of c. 80 000 3′-end spruce ESTs are provided adjacent to colour bars, along with expect values that represent BLASTX scores to The Arabidopsis Information Resource (TAIR) Arabidopsis thaliana peptide set or published conifer protein sequences [terpene synthases (TPS) and cytochrome P450s]. The ‘+’ or ‘−’ signs within boxes indicate statistical significance (P-value < 0.05) of up- or down-regulation, respectively. ESTs marked with an asterisk were also examined by real-time PCR (Fig. 7 and Table 3). A complete list of spruce ESTs with ID numbers listed in the same order (i.e. grouped by enzyme, top to bottom) as they appear in the figure is provided in Supplementary Table S3. AACT, acetyl-CoA acyl transferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MPDC, mevalonate diphosphate decarboxylase; DXPS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase; MECPS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; gcpE, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; lytB, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IPPI, isopentenyl diphosphate: dimethylallyl diphosphate isomerase; GPPS, geranyl diphosphate synthase; FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase.
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Figure 6. Metabolic pathway scheme for phenylpropanoid metabolism. Arrows represent enzymatic reactions and boxes represent metabolic products. Each row of four coloured boxes adjacent to arrows represents an expressed sequence tag (EST) on the spruce 9.7K array, with individual boxes corresponding to the relative fold-change between a treatment and control for each type of treatment from left to right: mechanical wounding of bark 24 h, weevil feeding on bark 48 h, budworm feeding of green shoot tips 3 h, budworm feeding of green shoot tips 52 h. The colour scale indicates fold-change differences in gene expression between treated and control samples. Contig (c) numbers derived from a CAP3 sequence assembly of c. 80 000 3′-end spruce ESTs are provided adjacent to colour bars, along with expect values that represent BLASTX scores to The Arabidopsis Information Resource (TAIR) Arabidopsis thaliana peptide set or published conifer protein sequences [chalcone (CHS) and stilbene (STS) synthases]. The ‘+’ or ‘–’ signs within boxes indicate statistical significance (P-value < 0.05) of up- or down-regulation, respectively. ESTs marked with an asterisk were also examined by real-time PCR (Fig. 7 and Table 3). A complete list of spruce ESTs with ID numbers listed in the same order (i.e. grouped by enzyme, top to bottom) as they appear in the figure is provided in Supplementary Table S3. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; C3H, p-coumarate-3-hydroxylase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase; COMT, caffeic acid O-methyltransferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; 4CL, 4-coumarate-CoA ligase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; F5H, ferulate-5-hydroxylase; DIR, dirigent.
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Figure 2. Overall changes of gene expression induced by weevils, budworms, or mechanical wounding. Venn diagrams showing distinct and overlapping patterns of genes induced or repressed by weevil feeding (48 h), budworm feeding (3 and 52 h), or mechanical wounding (24 h). All genes identified were differentially expressed (fold-change > 1.5x and P-value < 0.05) between treatment and control RNA samples. (a & b) Intersection of genes that were up-regulated (a) or down-regulated (b) between budworm feeding after 3 and 52 h. (c & d) Intersection of genes that were up-regulated (c) or down-regulated (d) between weevil feeding, budworm feeding (52 h) and mechanical wounding.
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Overall similarities and differences in the spruce host transcriptome response to insect herbivory and wounding
Considerable overlap was observed between the responses to weevil or budworm herbivory and mechanical wounding, with 770 DE elements in common, despite the fact that different spruce tissues were compared (Fig. 2c & d). The overlap of DE elements was greatest between weevil feeding and mechanical wounding in bark tissue (1586 elements), with less overlap between the responses induced by the two insects feeding in different tissues, shoot tips and bark (1149 elements), or between mechanical wounding in bark and budworm feeding in shoot tips (1089).
To further analyse and illustrate the temporal patterns to budworm feeding in shoot tips and to compare weevil feeding and mechanical wounding in bark tissue, we generated scatter plots of log2 treatment : control expression ratios. Array elements were classified as commonly or uniquely DE between treatments by dividing the plots into subplanes and incorporating 95% confidence intervals for the expression of each element as a statistical measure of the likelihood that an array element belongs within a subplane (Fig. 3). Using a minimum fold-change criteria of 1.5x and 3x, respectively, for budworm herbivory at 3 and 52 h, we identified 64 array elements DE at both time points, 47 elements DE only at 3 h and 387 elements DE only at 52 h. To compare responses in bark to weevil feeding and mechanical wounding, we applied a twofold-change criteria and observed 339 array elements DE in response to either treatment, 361 elements DE only after wounding and 55 elements DE only after weevil feeding.
Figure 3. Relative changes in gene expression (a) in green shoot tips 3 h and 52 h after the onset of budworm (BW) feeding and (b) in bark 24 h after mechanical wounding and 48 h after the onset of weevil feeding. Expression ratios are presented as log2 fold-change between treated and control (C) plants (see Materials and Methods). The left and right scatter plots are identical, except that genes identified as differentially expressed (DE) between treatments are shaded in colour in the right-hand panels. In panel (a), red dots represent genes that are DE at both 3 h and 52 h; green dots represent genes DE after 3 h, but not after 52 h; and blue dots represent genes DE after 52 h, but not after 3 h. In panel (b), red dots represent genes DE after both mechanical wounding and weevil feeding; green dots represent genes DE after mechanical wounding, but not weevil feeding; blue dots represent genes DE after weevil feeding, but not mechanical wounding; and magenta dots represent a group of genes that are down-regulated by weevil feeding, and are contained within a distinct cloud of data points. The same colour coding is used in Table 2 to indicate the association of individual genes with expression groups identified here. Subplane divisions were selected to capture the upper ∼ 15% of genes that are most DE relative to control trees for each treatment. In both panels, black dots on the right-hand scatter plot represent genes that showed no DE or whose unadjusted 95% confidence intervals were not completely contained within the subplane.
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Functional classification of insect- and wound-induced transcripts in Sitka spruce
Functional annotation of conifer genes is constrained by the often limited sequence similarity of genes from gymnosperms and angiosperms, and that the majority of annotated plant genes in public databases are from angiosperms. Nevertheless, Arabidopsis gene annotation databases (e.g. TAIR; http://www.arabidopsis.org) provide a useful common reference system for initial functional classification. Biotic stress- and wound-induced genes identified in our microarray analysis were classified into 10 functional groups based on annotation to Arabidopsis, and the most prominent genes are shown in Table 2. Viewed broadly, they include genes involved in general metabolism, photosynthesis, transport, transcriptional regulation, response to stress, detoxification and redox processes, octadecanoid and ethylene signalling, secondary metabolism, as well as cDNAs with no significant match to Arabidopsis and genes of uncharacterized biological processes. In the following sections we will briefly highlight some of the findings of insect-induced gene responses for each of the 10 functional categories. A specific emphasis will be on defence gene systems of special relevance for conifers, particularly signalling and responses in the terpenoid and phenolic secondary metabolite pathways. We will also briefly compare the transcriptome response to insect herbivory in this gymnosperm tree system with a very similar recent microarray analysis of plant-insect responses in an angiosperm tree species, poplar (Populus deltoides × trichocarpa), responding to foliage feeding forest tent caterpillars (Ralph et al. 2006a).
Genes of uncharacterized biological function affected by insect herbivory or wounding
Examples of microarray cDNA elements with no similarity to Arabidopsis that represent insect- or wound-responsive genes include IS0011_A15, WS0044_F23, WS0034_E23 and WS0044_G09 (Table 2). These ESTs have high similarity to other spruce ESTs in our sequence database, as well as to gymnosperm EST sequences in the public domain, confirming that they represent true conifer genes. In addition, several spruce ESTs with similarity to Arabidopsis genes are annotated as uncharacterized biological function. Examples include a photoassimilate-responsive protein (WS0014_J09) selectively induced after budworm feeding, a lateral organ boundaries (LOB) protein (IS0014_J19) induced after weevil feeding, and a phosphate-responsive protein (WS0015_F13) and a lipase containing the GDSL amino acid motif (WS0016_C06), both strongly induced by budworm feeding (Table 2). Spruce EST WS0014_J09 is weakly similar (TBLASTX; E 1e−07) to the PAR-1a and PAR-1b transcripts from tobacco that are induced in response to high soluble sugar levels in leaves, salicylate and after infection with potato virus Y (Herbers et al. 1995). LOB proteins are expressed in the boundary found between lateral organs and shoot apical meristems and are presumably involved in plant development (Shuai, Reynaga-Pena & Springer 2002). Similarly, spruce ESTs of uncharacterized function down-regulated in bark tissue in response to mechanical wounding or weevil feeding included plantacyanin (WS0018_A07), belonging to the phytocyanin family of blue copper proteins with a proposed role in reproduction (Dong, Kim & Lord 2005), and auxin-regulated (WS0038_N22) and low temperature and salt responsive (WS0017_F02) proteins.
Genes of general metabolism affected by insect herbivory or wounding
We identified several genes involved in general metabolism that were consistently induced by wounding and both insect treatments including carbonic anhydrase (WS00110_A15), an enoyl-CoA hydratase (WS00113_F20), and an acid phosphatase (IS0011_B10) (Table 2). During photosynthesis in C4 plants, carbonic anhydrase is involved in converting CO2 into bicarbonate for fixation by the primary carboxylating enzyme phosphoenolpyruvate carboxylase. A carbonic anhydrase in tobacco was shown to bind salicylic acid in chloroplasts, have antioxidant activity and function in the hypersensitive response in plant disease resistance (Slaymaker et al. 2002). Enoyl-CoA hydratase is potentially involved in a broad array of β-oxidation processes to degrade fatty acids.
Other array elements for genes involved in general metabolism were induced only in shoot tips after budworm feeding including an xyloglucan endo-1,4-β-D-glucanase (WS0038_G12), which catalyses the endo-hydrolysis of β-1,4-glucan linkages in cellulose and xyloglucan in plant cell walls; an iron-sulphur complex protein (WS0013_D08), which are found in both mitochondria and plastids of plants and are essential for respiration and photosynthesis (Léon et al. 2003); a very long chain fatty acid (VLCFA)-condensing enzyme (WS00113_A19), which controls the rate of VLCFA biosynthesis important for waxy cuticle formation (Millar et al. 1999); and a calcium-binding EF hand protein (WS0016_L07), which have previously been demonstrated to be induced after insect feeding in leaves of Arabidopsis (Stotz et al. 2000), and are likely to have a regulatory role in the plant response rather than direct defensive function because members of this superfamily are involved in a wide variety of calcium-regulated cellular processes (Day et al. 2002).
There were also a large number of genes involved in energy and other primary metabolisms strongly down-regulated after budworm feeding that were generally less responsive to weevil feeding or wounding in bark (Table 2). These include two steps in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (WS00110_E02) and fructose-1,6-biphosphate aldolase (WS0018_O18); two steps in the Calvin cycle, fructose-1,6-biphosphatase (WS0019_N05) and transketolase (WS0041_E18); galactinol synthase (WS0013_N23), responsible for raffinose family oligosaccharide production; hydroxypyruvate reductase (WS0015_L24) of the photorespiratory carbon oxidation cycle; glutamine synthase (WS0015_E01), which combines ammonium with glutamate to form glutamine during nitrogen fixation; and cellulose synthase (WS0039_G18), which transfers a glucose residue to glucan chains in the cellulose microfibrils of cell walls. These differences between the response in induced bark and shoot tips could be a result of higher constitutive gene expression, combined with insect-induced redirection of energy and primary metabolisms in green shoot tips and young needles, which may be less important in the bark tissues.
Transport genes affected by insect herbivory or wounding
Plant defences against insects are likely to involve regulation of transport processes both for local and systemic defence signalling, as well as for the biosynthesis and local accumulation of defence chemicals. Among spruce genes associated with transport functions, we identified an oligopeptide transporter (WS0042_C02) strongly induced in bark after wounding or weevil feeding (Table 2), which presumably translocates small peptides across cellular membranes in an energy dependent manner. The physiological role of peptide transport is still undefined; possible substrates for these transporters include glutathione, γ-glutamyl peptides, hormone-amino acid conjugates, peptide phytotoxins, and oligopeptides such as systemin with apparent signalling function in the induction of a systemic defence response to wounding by herbivores (McGurl et al. 1992; Stacey et al. 2002). Likewise, an ATP-Binding Cassette (ABC) protein (IS0011_I18) was also induced by both treatments in bark. In addition to their traditional role in detoxification processes in animal cells, ABC proteins in plants have been demonstrated to participate in chlorophyll biosynthesis, formation of Fe/S clusters, stomatal movement and ion fluxes (Martinoia et al. 2002). ABC proteins may also be directly involved in plant defence via transport of signalling molecules such as jasmonate (Theodoulou et al. 2005), transport of phytochemicals as has been shown for alkaloid (Shitan et al. 2003) and terpenoid (Jasiński et al. 2001) defence compounds, or reinforcement of cuticular waxes (Pighin et al. 2004). A large number of putative transport proteins, including several ABC proteins, were also induced in poplar in response to feeding by defoliating forest tent caterpillars (Ralph et al. 2006a), further supporting the importance of transport mechanisms in plant defence against insects.
One of the more abundant classes of DE genes involved in transport/general metabolism is the lipid transfer proteins (LTP). We observed some LTP genes to be induced by all three treatments, other LTPs were repressed by all three treatments, and others were selectively induced by budworm feeding only. LTPs are small, basic proteins synthesized as precursors that transfer phospholipids between membranes, bind fatty acids in vitro, and have been proposed to be associated with plant–insect interactions by contributing to cutin biosynthesis (Kader 1996), pathogen-defence reactions (Garcia-Olmedo et al. 1995), and the recognition of intruders in plants and in systemic resistance signalling (Blein et al. 2002; Maldonado et al. 2002). The interpretation of function is complicated by the fact that LTPs are represented by a large number of genes with several subfamilies, and thus far no systematic characterization of LTPs has been performed in any plant species.
Transcriptional regulation affected by insect herbivory or wounding
Transcriptional regulation and intracellular signalling cascades for plant defence, including induced secondary metabolism, are poorly understood. Microarray expression profiling could be a useful approach to identify signalling and regulatory events in the induction of plant defence against insects as previously shown for Arabidopsis and poplar (De Vos et al. 2005; Ralph et al. 2006a). Weevil feeding and wounding in bark activated genes from the basic helix-loop-helix (bHLH; IS0013_F06) and bZIP (WS0044_J05) transcription factor families, and budworm feeding activated a member of the MADS-box transcription factor family (WS0042_F06) (Table 2). Each of these transcription factor classes is represented by large gene families in Arabidopsis (i.e. bHLH, 139 genes; bZIP, 81 genes; MADS-box, 82 genes; Riechmann et al. 2000). To date most transcription factors linked to plant stress responses have been derived from the AP2/EREBP, WRKY, MYB and bZIP families (Stracke, Werber & Weisshaar 2001; Singh, Foley & Oñate-Sánchez 2002). The fact that we identified only a small number of DE transcription factors in the present study likely reflects their paucity of representation on the microarray. To address this issue in future work, we have recently constructed a second, larger spruce cDNA microarray containing a larger number of transcription factors identified in normalized spruce cDNA libraries (S. Ralph & J. Bohlmann, unpublished data).
General stress response genes affected by insect herbivory or wounding
Among genes commonly associated with plant responses to biotic stress, we observed several spruce lectin proteins to be selectively induced in bark after wounding (e.g. IS0012_N03), and β-1,3-glucanase genes to be induced in either bark (WS0017_B07) or shoot tips (WS0021_I16) (Table 2). Lectins are carbohydrate-binding proteins, many of which have insecticidal activity (Peumans & Van Damme 1995). β-1,3-glucanases are pathogenesis-related (PR) proteins that are rapidly induced during fungal invasion and are proposed to contribute to plant defence by digesting cell wall components of the fungal pathogen. In white spruce, β-1,3-glucanase genes have previously been shown to be inducible in response to wounding, drying and flooding stresses, along with treatment with Leptosphaeria maculans fungal extract (Dong & Dunstan 1997). Chitinases were also induced in response to insect feeding and wounding in spruce bark (e.g. IS0013_J03). Chitinases represent a large and diverse group of enzymes that catalyse the cleavage of internal β-1,4-glycoside bonds present in the biopolymers of N-acetylglucosamine found in chitin, a major component of fungal, bacterial and insect cell walls. Plant chitinases are classified as PR proteins and have been demonstrated in many plant systems, including spruce (Dong & Dunstan 1997; Hietala et al. 2004), to be transcriptionally induced in response to both biotic (e.g. viruses, bacteria, fungi, insect pests) and abiotic (e.g. drought, salinity, wounding, plant hormones) stress (Kasprzewska 2003). There is also emerging evidence that chitinase-like proteins may play a role in normal plant growth and development, as demonstrated by analysis of the AtCTL1 chitinase-like gene responsible for the elp1 mutation in Arabidopsis (Zhong et al. 2002). Several late embryogenesis abundant (LEA) genes, also known as dehydrins (e.g. IS0013_A20) were also strongly induced by insects and wounding in Sitka spruce shoot tips and bark. LEA proteins are also induced in plant responses to other environmental stresses such as drought, salinity and freezing, and are found in high abundance during the late stages of embryogenesis; however, their physiological function is unclear (Wise & Tunnacliffe 2004). A LEA/dehydrin transcript from white spruce has previously been demonstrated to be induced in response to wounding, MeJA, drought, cold and abscisic acid (Richard et al. 2000). Glucanases, chitinases, lectins and dehydrins were also commonly induced in poplar leaves after feeding by forest tent caterpillars (Ralph et al. 2006a); however, the large number of strongly induced protease inhibitor class transcripts observed in poplar were generally absent in spruce tissues, suggesting this class of proteinaceous defences may not be utilized in spruce.
Detoxification and redox processes affected by insect herbivory or wounding
Tissue damage imposed by herbivore feeding is known to cause oxidative stress. Insect herbivory can expose plant cells to potentially toxic or otherwise biologically active metabolites, such as components of insect saliva or host secondary metabolites that are normally restricted to specialized cells or subcellular compartments. It therefore is not surprising that several genes potentially involved in protection of cells from oxidative stress or involved directly in detoxification were induced by one or more insect or wound treatments. These genes include, for example, lactoylglutathione lyase (WS0017_M23), also known as glyoxalase I, peroxidases (e.g. WS00110_M24), thioredoxin (e.g. WS0034_I08), cytochrome b5 (WS0021_L21), oxidoreductases (e.g. WS0041_G15) and cytochrome P450s (e.g. IS0012_L15) (Table 2). Lactoylglutathione lyase catalyses the first step in the glyoxalase system for the glutathione-based detoxification of methylglyoxal, which is formed primarily as a by-product of carbohydrate and lipid metabolism. In addition, this enzyme was demonstrated to be induced in response to drought and cold stresses in Arabidopsis (Seki et al. 2001). Peroxidase enzymes are associated with the oxidation of phenolic compounds in cell walls and the polymerization of lignin and suberin, and in spruce have been demonstrated to be induced in response to infection by the pathogen Pythium dimorphum (Fossdal et al. 2001). Not surprisingly, many of the same detoxification genes induced in spruce after insect feeding or wounding were also observed in poplar leaves after forest tent caterpillar feeding (Ralph et al. 2006a), suggesting there is a common set of genes involved in the oxidative stress response.
Octadecanoid and ethylene pathway genes affected by insect herbivory or wounding
Plant responses to biotic and abiotic stresses are regulated locally and systemically by a complex network of signalling cascades including peptide signals (e.g. systemin), salicylic acid, ethylene, H2O2, and fatty acid-derived oxylipins such as the octadecanoid-derived jasmonic acid (JA) and MeJA (Howe 2004; Halitschke & Baldwin 2005). Earlier work has established that MeJA, mechanical wounding and insects can trigger similar traumatic resin defence responses in conifers (Martin et al. 2002, 2003; Hudgins et al. 2003; Miller et al. 2005). In addition, Miller et al. (2005) have shown up-regulation of putative allene oxide synthase (AOS) and allene oxide cyclase (AOC) transcripts in spruce after weevil feeding and MeJA application. Furthermore, Hudgins & Franceschi (2004) showed that MeJA-induced development of traumatic resin ducts is induced via ethylene signalling. We therefore evaluated representation of genes of the octadecanoid and ethylene pathways on the spruce 9.7K cDNA microarray via similarity searches against known genes from Arabidopsis (Turner, Ellis & Devoto 2002; Stenzel et al. 2003) using a combination of BLASTX and TBLASTN searches of the TAIR Arabidopsis peptide set with a stringent threshold (E < 1e−20) (Table 2 and Supplementary Table S3). The formation of MeJA from membrane lipids in the octadecanoid pathway involves seven enzymatic steps [i.e. phospholipase, lipoxygenase (LOX), AOS, AOC, 12-oxo-phytodienoic acid reductase, β-oxidization enzymes and JA methyl transferase (Howe 2004)], of which six are represented on the spruce 9.7K microarray by 14 ESTs (Fig. 4; Supplementary Table S3). The first three steps in this pathway can also lead to 6-carbon alcohols, which require two additional enzymes (hydroperoxide lyase and alcohol dehydrogenase, the latter represented by two candidate ESTs on the array). Within the octadecanoid pathway several ESTs representing genes encoding proteins with high similarity to LOX, AOS and AOC (e.g. LOX, IS0014_J20; AOS, WS0023_N05; AOC, WS0039_A09; Table 2) were up-regulated with both weevil and budworm feeding and with mechanical wounding treatments (Fig. 4 and Table 2). The induction of LOX, AOS and AOC transcripts in Sitka spruce in response to insect feeding and wounding, in different tissues, supports that these enzymes, and jasmonates in general, are important in activating and/or modulating the spruce defence response.
Two genes potentially involved in ethylene biosynthesis were also induced by both insect species and wounding in bark and shoot tissues, S-adenosylmethionine synthase (SAM synthase, WS0035_O19) and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase, WS00112_P16) (Table 2). Ethylene is an important modulator in defence signal transduction (Feys & Parker 2000) that has been demonstrated to be induced in response to insect herbivory in several plant systems (Arimura et al. 2000; Winz & Baldwin 2001), as well as wounding in conifers (Hudgins et al. 2006), and has been demonstrated to regulate defence-oriented genes such as protease inhibitors (O’Donnell et al. 1996), defensin (Penninckx et al. 1998) and PR proteins (Díaz, ten Have & van Kan 2002). Building on the findings of Hudgins & Franceschi (2004), the present EST and microarray analysis of spruce candidate genes for ethylene and octadecanoid formation will allow a more detailed analysis of spatial and temporal signalling activities in spruce defence against insects.
Terpenoid secondary metabolism genes affected by insect herbivory or wounding
Terpenoids are formed in large volumes in conifer oleoresin and are perhaps the most prominent constitutive and inducible chemical defences (for recent reviews, Bohlmann & Croteau 1999; Phillips & Croteau 1999; Trapp & Croteau 2001; Huber et al. 2004; Martin & Bohlmann 2005). The pathways for isoprenoid biosynthesis lead to the production of hundreds of terpenoid compounds that are the major components of oleoresin defences and volatile emissions in conifers (Fig. 5). The formation of terpenoids involves two pathways for the formation of isoprenoids, the mevalonic acid (MEV) and the methylerythritol phosphate (MEP) pathways, followed by a series of condensation reactions catalysed by prenyl transferases, namely geranyl diphosphate (GPP) synthase, farnesyl diphosphate (FPP) synthase and geranylgeranyl (GGPP) diphosphate synthase (Lange & Ghassemian 2003). GPP, FPP and GGPP are converted by TPS genes to the many basic mono-, sesqui- and diterpenoid structures found in conifers (Martin et al. 2004). Additional modification and structural diversification of terpenoids involves cytochrome P450 dependent monooxygenases, such as in the formation of diterpene resin acids (Ro et al. 2005).
Induced terpenoid resin defences and induced volatile emissions in spruce have been well characterized at the anatomical, biochemical and molecular levels for the role of a family of TPS (Martin et al. 2002, 2003, 2004; Byun-McKay et al. 2003, 2006; Fäldt et al. 2003; Miller et al. 2005), while the majority of other enzymes in these pathways have not yet been adequately investigated in insect-induced terpenoid defence responses in conifers. To address this issue, we evaluated representation of genes of the isoprenoid pathway on the spruce 9.7K cDNA microarray (Fig. 5; Supplementary Table S3) via similarity searches against known genes in Arabidopsis and conifers beginning from the first step of the MEP pathway (i.e. 1-deoxy-D-xylulose 5-phosphate synthase) to candidates for the final cytochrome P450 modification of terpenoid products (Lange & Ghassemian 2003; Martin et al. 2004; Ro et al. 2005) using a combination of BLASTX and TBLASTN searches of the TAIR Arabidopsis peptide set and the nonredundant (nr) division of GenBank with a stringent threshold (E < 1e−20). Among the 13 early steps of isoprenoid biosynthesis leading to the production of isopentenyl diphosphate or dimethylallyl diphosphate via the MEV pathway in the cytosol and endoplasmic reticulum or the MEP pathway in plastids, seven enzymatic steps are represented by 12 ESTs on the 9.7K array (Fig. 5; Supplementary Table S3). Of these, up-regulation was observed for 3-hydroxy-3-methylglutaryl-CoA synthase, mevalonate diphosphate decarboxylase and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase by weevil feeding and mechanical wounding in bark tissues, as well as for 3-hydroxy-3-methylglutaryl-CoA synthase and reductase, and the lytB ortholog by budworm feeding (Fig. 5). Prior to the action of the prenyl transferases there is up-regulation of an EST representing isopentenyl diphosphate isomerase by weevil feeding and mechanical wounding in bark (Fig. 5). Among the six putative GGPP synthase ESTs represented on the array (no ESTs with similarity to FPP or GPP synthases were identified), ESTs from two distinct contigs (1499 and 3816) were strongly up-regulated by both insect species and by mechanical wounding, suggesting some differential insect-induced expression for members of this GGPP synthase gene family (Fig. 5; also see GGPP synthase WS00111_A06 and WS00111_K11 in Table 2). A large number of known, as well as some putatively new TPS genes (23 ESTs) are spotted on the spruce 9.7K array and the vast majority are induced, often strongly, in response to each type of insect feeding or wound treatment (Fig. 5, also see selected TPS in Table 2). Although subtle differences in expression among TPS genes is apparent, the high nucleotide similarity among spruce TPS family members (Martin et al. 2004) limits the ability to distinguish between gene-specific expression and cross-hybridization between closely related family members. Finally, among nine ESTs with similarity to four cytochrome P450 genes from loblolly pine, one of which catalyses a series of consecutive oxidation steps with different diterpenol and diterpenal intermediates (Ro et al. 2005), two ESTs were induced by weevil feeding or wounding in bark, as well as budworm feeding in leaders after 52 h. This first comprehensive microarray expression analysis of insect-induced conifer terpenoid pathway genes identified new targets for characterization both in the early and intermediate steps of the pathway, as well as new candidates for functional analyses of cytochrome P450 enzymes in terpenoid defences.
Phenolic secondary metabolism genes affected by insect herbivory or wounding
In addition to terpenoids, phenolic secondary metabolites have been proposed to play a role in conifer defence against pathogens and potentially insects as well (Brignolas et al. 1995; Huber et al. 2004; Franceschi et al. 2005; Ralph et al. 2006b). However, much less is known about the molecular regulation of this pathway in conifer insect defence compared to the formation of terpenoids. Phenylpropanoid metabolism builds on the shikimate pathway, which links the metabolism of carbohydrates to the biosynthesis of aromatic amino acid precursors, phenylalanine and tyrosine, in the formation of phenolic compounds. In a series of seven metabolic steps, phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) are converted to chorismate, which is the precursor of the aromatic amino acids tryptophan, phenylalanine and tyrosine (Herrmann & Weaver 1999). Spruce genes representing two enzymatic steps within the shikimate pathway were induced by wounding and both types of insect feeding in bark and green shoot tips: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (WS0038_H08), the first step involving condensation of PEP and E4P; and shikimate kinase (WS00112_G07), which is the fifth step of the pathway that catalyses the phosphorylation of shikimate to yield shikimate-3-phosphate (Table 2).
Following the formation of aromatic amino acids in the shikimate pathway, most secondary phenolic compounds, such as flavonoids, stilbenoids, condensed tannins and other polyphenolics along with the structural polymer lignin, are subsequently derived from phenylalanine via the action of a complex metabolic grid of enzyme activities (Dixon et al. 2001). We evaluated representation of the core phenylpropanoid pathway and select steps of branch pathways on the spruce 9.7K cDNA microarray (Fig. 6; Supplementary Table S3) via similarity searches against known genes from Arabidopsis (Costa et al. 2003; Hoffmann et al. 2003; Raes et al. 2003) and conifers (Kodan, Kuroda & Sakai 2002; Ralph et al. 2006b) using a combination of BLASTX and TBLASTN searches of the TAIR Arabidopsis peptide set and the nr division of GenBank with a stringent threshold (E < 1e−20). There are presently 10 known enzymes for the monolignol biosynthesis pathway [i.e. phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), p-coumarate-3-hydroxylase (C3H), ferulate-5-hydroxylase (F5H), 4-coumarate CoA ligase (4CL), caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD)], most of which are represented by small gene families in Arabidopsis that collectively encode 18 genes for which there is evidence of involvement in phenylpropanoid and lignin biosynthesis (Raes et al. 2003; Ehlting et al. 2005). In addition, as many as 45 additional genes annotated as related to phenylpropanoid genes encode enzymes of unknown specific function (Costa et al. 2003; Raes et al. 2003). On the spruce 9.7K cDNA microarray we identified 81 ESTs representing eight of the 10 enzymes (there were no representative ESTs for F5H or HCT).
At the entry point to phenylpropanoid metabolism the enzymes PAL and C4H are represented by 10 and seven ESTs, respectively, all of which showed up-regulation by at least one form of insect feeding or wounding (Fig. 6, e.g. PAL, IS0011_E16; and C4H, WS0031_M22 in Table 2). Among other genes from subsequent steps along the phenylpropanoid pathway, array elements for C3H, 4CL, COMT and CCoAOMT all demonstrate up-regulation for at least some members of each gene family (Fig. 6, e.g. 4CL, WS0031_C20; COMT, WS0021_F13; and CCoAOMT, WS0041_M02 in Table 2). However, there are also notable differences among gene family members for several enzymatic steps such as among the CCoAOMT genes where ESTs representing contigs 7294 and 494 demonstrate consistent up-regulation in response to insect feeding and mechanical wounding (Fig. 6), whereas ESTs representing CCoAOMT contig 489 are down-regulated in the same bark tissues, and ESTs representing CCoAOMT contigs 8234 and 30 are only DE after budworm feeding for 52 h. In each case, the expression data among multiple ESTs representing each gene is remarkably consistent. Further annotation of spruce phenylpropanoid gene families encoding enzymes such as 4CL, COMT and CCoAOMT using FLcDNA sequences and comparative genomics will be required to determine if all such genes on the array encode bona fide phenylpropanoid enzymes (S. Ralph & J. Bohlmann, unpublished data). For example, of the seven Arabidopsis genes annotated as encoding enzymes related to CCoAOMT, only one has an expression pattern consistent with a function in monolignol biosynthesis (Ehlting et al. 2005). Thus, the spruce CCoAOMT ESTs with divergent expression patterns could represent CCoAOMT-like genes with functions distinct from true CCoAOMTs. Studies using gene-specific primers and real-time PCR will also be required to dissect complex microarray profiles in gene families such as CCoAOMT, in which individual gene family members may be differentially regulated. Two other phenylpropanoid enzymes represented on the array, CCR and CAD, show limited differential expression except for ESTs representing CAD contig 1113 (Fig. 6), suggesting that carbon flux into monolignols themselves may not be a primary response to mechanical wounding and herbivory.
We also observed several other genes for enzymes of monolignol biosynthesis and other branches of phenylpropanoid metabolism to be induced after insect feeding or wounding, including laccases/diphenol oxidases (e.g. WS0016_N04), DIR proteins (e.g. IS0013_K10), and genes of flavonoid metabolism (e.g. chalcone synthase, IS0013_L11; chalcone isomerase, WS0011_B22; anthocyanin 5-aromatic acyltransferase, WS00110_P10; flavonoid 3-hydroxylase, WS0022_G09; leucoanthocyanidin dioxygenase, WS0014_M03; and dihydroflavonol 4-reductase, WS0016_K11) (Fig. 6 and Table 2).
Laccases are proposed to be involved in the polymerization of monolignols to produce lignin and lignans based on their ability to oxidize monolignols and their close spatial and temporal correlation with lignin deposition (Boerjan, Ralph & Baucher 2003). Using a set of 17 Arabidopsis laccase (diphenol oxidase) search sequences collected from GenBank we identified 10 spruce ESTs spotted on the 9.7K cDNA microarray with high similarity (E < 1e−20; Fig. 6; Supplementary Table S3). ESTs from two contigs (6965 and 13 708) were strongly up-regulated in response to all treatments, whereas other laccase ESTs were only moderately up-regulated, if at all. Increased gene expression of laccases could lead to strengthening of cell walls during insect attack via increased lignin deposition and/or increased production of toxic lignans.
DIR proteins are proposed to direct, in the presence of a laccase or other oxidase, the stereospecific coupling of monolignols to form lignans, and possibly lignin (Davin et al. 1997). We have previously identified 19 putative DIR genes in spruce, several of which are rapidly inducible in bark in response to weevil feeding and wounding (Ralph et al. 2006b). A TBLASTN search using the 17 unique DIR FLcDNAs and two partial cDNAs from spruce identified 22 DIR-like ESTs spotted on the spruce 9.7K cDNA microarray (E < 1e−20; Fig. 6; Supplementary Table S3). The expression of these ESTs falls into two patterns, those up-regulated under all treatments (Fig. 6, i.e. contigs 7094, 128, 139 and 22) and those that are unresponsive to insect feeding or mechanical wounding in bark tissue, but that are up-regulated after budworm feeding for 52 h (Fig. 6, i.e. contigs 13 251, 2062, 6069, 720, 1223 and 8122). This is in agreement with our recent phylogenetic analysis and independent transcript expression analysis of spruce DIR gene family members, which suggest that these two groups of contigs belong to the DIR-a and DIR-b subfamilies, respectively, and only members of the spruce DIR-a subfamily appear to be induced in bark tissue after weevil feeding or mechanical wounding (Ralph et al. 2006b).
Finally, in the production of flavonoids/stilbenoids, the first step is catalysed by chalcone/stilbene synthase, which is frequently induced at the transcript level in various plant species, including spruce (Nagy et al. 2004b), in response to a variety of stresses (Dixon & Paiva 1995). Using chalcone and stilbene synthase search sequences from a variety of conifer species (Kodan et al. 2002; and additional sequences identified within GenBank) we identified 17 ESTs spotted on the spruce 9.7K cDNA microarray (E < 1e−20; Fig. 6; Supplementary Table S3); however, without functional testing it is not possible to reliably classify these ESTs as specifically representing a chalcone or stilbene synthase. A consistent pattern of up-regulation after insect feeding or wounding was observed among the majority of chalcone/stilbene synthase-like ESTs. The role of many of the insect-induced genes of the various branch points of spruce phenolic secondary metabolism remains to be investigated with regard to the enzymes’ specific contributions to generate individual defence metabolites, for example those associated with phenolic parenchyma cells that have not yet been chemically profiled (Franceschi et al. 2005). The present gene expression profiling of insect-induced genes of these branch pathways contributes targets for functional characterization.
Refined gene-specific expression using real-time PCR
In order to validate cDNA microarray expression profiles and obtain more refined gene expression data, we designed gene-specific primers for a subset of 18 transcripts selected from Table 2 because of their potential biological significance in plant defence and because collectively they exhibited both small and large fold-change DE between treatments (Fig. 7 and Table 3). Overall, very similar results (up- or down-regulation) were obtained for most transcripts between the two techniques of expression analysis, although the magnitude of the response was often greater with real-time PCR, likely as a result of the larger linear dynamic range of detection and use of gene-specific primers. Among the 18 transcripts examined, we observed excellent agreement between microarray and real-time PCR expression data for 15 transcripts including: AP2 (WS00111_M12) and bZIP (WS0044_J05) transcription factors, chitinase (IS0013_J03), ABC transport protein (IS0011_I18), ferredoxin WS0024_M05, LOX (IS0014_J20), AOC (WS0039_A09), TPS (WS0017_E16), PAL (IS0011_E16), C4H (WS0031_M22), chalcone/stilbene synthase (IS0013_L11), dihydroflavonol 4-reductase (WS0016_K11), DAHP synthase (WS0038_H08), laccase (IS0011_E08) and a DIR transcript (IS0013_K10). For three of the transcripts examined we did observe differences in expression determined using these two methods (e.g. GGPPS, WS00111_A06; DIR, WS0031_H14; laccase, WS0016_N04). This suggests that cross-hybridization between closely related gene family members may complicate our interpretation of microarray results in some cases.
Figure 7. Relative abundance of 18 representative mRNA transcript species in green shoot tips subjected to budworm herbivory (B) 52 h after the onset of treatment, or bark tissue subjected to mechanical wounding (M) or weevil herbivory (W) 24 h and 48 h, respectively, after the onset of treatment. Control (C) shoot tip tissue (52 h) and bark tissue (24 h) were treated with Tween (Martin et al. 2002), and control bark tissue (48 h) received no treatment. Values represent mean ± SEM (n = 3 or more independent technical replicates) normalized to elongation factor 1α expression in each tissue. A Student’s t-test (two-sample, unpaired, one-sided) was performed to test significance of up- or down-regulation of each transcript between treated and control tissues (*P < 0.05; **P < 0.001). AOC, allene oxide cyclase; LOX, lipoxygenase; PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; DAHP synthase, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; CHS, chalcone/stilbene synthase; DFR, dihydroflavonol 4-reductase; LAC, laccase; DIR, dirigent; GGPPS, geranylgeranyl diphosphate synthase; TF, transcription factor; ABC, ATP-Binding Cassette protein; CHI, chitinase; FER, ferredoxin.
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Table 3. Relative abundance of 18 mRNA transcripts determined by real-time PCR
|Clone ID||BLASTX versus Arabidopsis||Mechanical at 24 h||Weevil at 48 h||Budworm at 52 h|
|WS0039_A09||Allene oxide cyclase|| 4.58|| 0.003|| 34.11|| 0.005|| 7.38|| 0.005|
|IS0014_J20||Lipoxygenase|| 9.32||< 0.001|| 10.63|| 0.001|| 5.21|| 0.002|
|IS0011_E16||Phenylalanine ammonia lyase|| 44.56||< 0.001|| 78.12||< 0.001|| 18.07||< 0.001|
|WS0031_M22||Cinnamate-4-hydroxylase|| 13.53||< 0.001|| 9.33||< 0.001|| 2.62|| 0.001|
|WS0038_H08||DAHP synthase|| 4.43|| 0.001|| 4.34||< 0.001|| 5.00|| 0.016|
|IS0013_L11||Chalcone/stilbene synthase|| 1.76|| 0.277|| 4.99|| 0.097|| 1.30|| 0.341|
|WS0016_K11||Dihydroflavonol 4-reductase|| 2.31|| 0.004|| 3.12|| 0.002|| 1.99|| 0.003|
|IS0011_E08||Laccase||595.97|| 0.009||2638.46|| 0.006||564.74|| 0.020|
|WS0016_N04||Laccase|| 0.47|| 0.041|| 1.05|| 0.481|| 0.29|| 0.041|
|WS0031_H14||Dirigent|| 0.22|| 0.151|| 1.28|| 0.377|| 0.34|| 0.002|
|IS0013_K10||Dirigent|| 70.77|| 0.002|| 19.50||< 0.001|| 6.95|| 0.002|
|WS00111_A06||Geranylgeranyl diphosphate synthase|| 0.98|| 0.475|| 3.49|| 0.099|| 0.87|| 0.352|
|WS0017_E16||Terpene synthase|| 27.67|| 0.002|| 8.90||< 0.001|| 12.06|| 0.005|
|WS0044_J05||bZIP transcription factor|| 3.27|| 0.054|| 5.67|| 0.006|| 1.49|| 0.259|
|WS00111_M12||AP2 transcription factor|| 7.11|| 0.029|| 8.50|| 0.039|| 2.34|| 0.018|
|IS0011_I18||ABC protein|| 23.95||< 0.001|| 34.42|| 0.004|| 13.68|| 0.001|
|IS0013_J03||Chitinase|| 26.63||< 0.001|| 131.96||< 0.001|| 14.82||< 0.001|
|WS0024_M05||Ferredoxin|| 0.31||< 0.001|| 0.50||< 0.001|| 0.56|| 0.003|