Combined transcript and metabolite analysis reveals genes involved in spider mite induced volatile formation in cucumber plants

Abstract

Many plants have an indirect defense against herbivores by emitting volatiles that attract carnivorous enemies of the herbivores. In cucumber (Cucumis sativus) the production of carnivore attractants can be induced by herbivory or jasmonic acid spraying. From the leaves of cucumber plants with and without spider mite infestation, two subtractive cDNA libraries were made that were enriched in cDNA fragments up- or down-regulated by spider mite infestation. A total of 713 randomly selected clones from these libraries were used to make a cDNA microarray. Subsequently, cucumber plants were sprayed with jasmonic acid, mechanically damaged, infested with spider mites, or left untreated (control). Leaf samples were taken at a range of different time points, and induced volatile compounds and mRNA (from the same leaves) were collected. cDNAs prepared from the mRNA were hybridized to the clones on the microarray. The resulting gene expression profiles were analyzed in combination with volatile production data in order to gain insight in the possible involvement of the studied genes in the synthesis of those volatiles. The clones on the microarray and the induced cucumber volatiles could be grouped into a number of clusters in which specific biosynthetic genes clustered with the product of that pathway. For example, lipoxygenase cDNA clones clustered with the volatile (Z)-3-hexenyl acetate and the volatile sesquiterpene (E,E)- alpha-farnesene clustered with an up-regulated sesquiterpene synthase fragment. This fragment was used to screen a cDNA library which resulted in the cloning of the cucumber (E,E)-alpha-farnesene and (E)-beta-caryophyllene synthases. The use of combined global gene expression analysis and metabolite analysis for the discovery of genes involved in specific biosynthetic processes is discussed.

Figure 1.

Score plot (85% of dynamics) of the overall difference in transcriptional behavior of clones printed on the chip after subtraction of average of columns and rows for the PCA. Data points are from SSH⁻- (black circles) and SSH⁺- (white circles) cDNA libraries. The more separated two plotted cDNA clones are, the more diverged is their gene expression profile.

Figure 2.

Volatile profile over time emitted from four leaf discs after spraying with 1 mm jasmonic acid (collected 0, 6, 24, 48, and 72 h after treatment; A), after spider mite infestation (collected 0, 6, 24, 48, 72, 96, and 168 h after treatment; B) or after spraying with 0.01% Tween 20 in water (C). In B, the right y axis represents data for (Z)-3-hexenyl acetate. Error bars indicate se of three replicates (A and B).

Figure 3.

Loading plot based on the overall gene expression in each of the different treatments. The transcriptional profile for each of the different treatments (columns) as two components explains 77% of the gene expression dynamics (PCA subtracting the average of columns and rows). The larger the distance between two sample-points the more diverged are the overall gene expression profiles for these treatments. Control (C), mechanically wounded (M), jasmonic acid sprayed (J), and spider mite infested (S). The number after each letter indicates hours after start of treatment.

Figure 4.

A, SOM with 24 components reflecting the gene expression and volatile formation patterns; PCA standardized rows, subtraction of average and subtraction of root mean square. Each circle or pie represents a cluster of genes with similar expression pattern. The larger the circle, the more clones in this particular group with a similar expression pattern. Areas where the intercircular space is dark indicate a similar expression profile between the neighboring clusters. The colored area inside the circles can be divided into wedge shaped pieces representing a specific gene or volatile. Intracircular color codes: Spider mite and early jasmonic-acid induced genes (beige), spider mite and late jasmonic-acid induced genes (green), (Z)-3-hexenyl acetate (yellow), (E)-β-ocimene (purple), 4,8-dimethyl-1,3(E),7-nonatriene (red), and (E,E)-α-farnesene (blue). cDNAs down-regulated by both spider mite infestation and jasmonic acid spraying are assigned to groups toward the A6 corner. B, Section of a dendrogram demonstrating (E,E)-α-farnesene production data clustering with four cDNAs, two of which are partial cDNA sequences of the (E,E)-α-farnesene synthase (5G8 and 8D11). Red describes high transcription/(E,E)-α-farnesene production rate and green (via black) color represents low transcription/emission rate. The 14 treatments of the cucumber plants are (left to right): control, 0 h; mechanical wounding, 6, 24, and 48 h; jasmonic acid spraying, 6, 24, 48, and 72 h; and spider mite infested, 24, 48, 72, 96, and 168 h and control 6 h.

Figure 5.

Logarithmic values for collected volatile emission data and cDNA expression data at the different treatments are plotted in the same diagram. (Z)-3-hexenyl acetate and lipoxygenase derived contigs 7, 8, 9, and 10 described in Table I (P ≤ 0.001; A). (E,E)-α-farnesene and cDNA fragments (5G8 and 8D11) from the cloned (E,E)-α-farnesene synthase gene (P ≤ 0.001; B). Dimethyl-1,3(E),7-nonatriene (DMNT) plotted with four peroxidase-like cDNA fragments putatively involved in their biosynthesis (P ≤ 0.001; C). (E)-β-ocimene together with 1-deoxy-d-xylulose 5-phosphate reductase-like cDNA fragment (6H4) (P ≤ 0.005) and the farnesene synthase cDNA fragments (P ≤ 0.001; D). Pearson correlation coefficient for the association of cDNA fragment to the volatile in that diagram is in brackets.

Figure 6.

GC-MS profiles of products formed by the heterologously expressed sesquiterpene synthase CsαFS and CsβCS with FDP as substrate. Chromatogram of product (m/z 93 + 161 + 189 + 204) obtained from assay with lysate of bacteria expressing recombinant CsαFS. The main product peak (Rt 13.95) is (E,E)-α-farnesene (A). Chromatogram of product (m/z 93 + 161 + 189 + 204) obtained from assay with lysate of bacteria expressing recombinant CsβCS. The main product peak (Rt 12.98) is (E)-β-caryophyllene by comparison with the Wiley GC-MS database (B). Chromatogram (m/z 93 + 161 + 189 + 204) of assay with lysate of bacteria expressing pET23c (empty vector; C). Mass spectrum of peak at 13.95 (D). Mass spectrum of peak at 12.98 (E). Compounds were identified by comparison with the Wiley GC-MS database, Adams (1995) and Joulain and König (1998).

Figure 7.

Alignment of the amino acid sequences of CsαFS and CsßCS with four sesquiterpene synthases catalyzing the formation of either (E,E)-α-farnesene (MxdαFS and PtαFS) or (E)-β̃-caryophyllene (AtβCS and AaβCS). MxdαFS and PtαFS are (E,E)-α-farnesene synthases from Malus × domestica (AAO22848) and Pinus taeda (AAO61226), respectively. AaβCS is an (E)-β̃-caryophyllene synthase from Artemisia annua (AAL79181) and AtβCS is an (E)-β-caryophyllene/α-humulene synthase from Arabidopsis (AAO85539). The RR-motif is indicated with ^**. The position of the cDNA fragments 8D11 and 5G8 (see text) is indicated.