Fig. 6: Oral secretions (OS) of Hi-1 mutant have no (3Z):(2E)-hexenal isomerase activity.
a Hexenal isomerase activity of OS from 5th instar wild-type (Hi-1+), heterozygous mutant (Hi-1+/-) and homozygous mutant (Hi-1-) larvae was determined by SPME-guided in vitro assay. Equal amounts (0.33 μg) of total protein per sample or a tenfold amount (3.3 μg) of homozygous mutant OS (labeled as Hi-1-(10x)) were incubated with Z-3-hexenal (0.2 mM). Buffer was used as control. The proportion of E-2-hexenal (in percentages) emitted from the total aldehyde headspace (Z-3-hexenal + E-2-hexenal) was calculated. Different letters indicate significant differences (p < 0.05) between tissues (One-way ANOVA followed by Tukey HSD post-hoc analysis, F4,23 = 57.97, p < 0.0001, n = 9 for buffer, n = 4 for Hi-1+, n = 5 for Hi-1+/-, Hi-1-, and Hi-1- (10x) biologically independent samples). b Representative extracted ion chromatograms (ion 69) as an output of the SPME-guided assay. The green highlight in E-2-hexenal peak area indicates a clear conversion of Z-3-hexenal to E-2-hexenal. c Emission of GLVs from leaf discs of N. attenuata. Leaf discs (2.4 mm diameter) were mechanically wounded and treated with 10 µL of Milli-Q water (w + water; n = 4 biologically independent samples), OS (10 µg total protein) from Hi-1+ (w + OS Hi-1+; n = 3 biologically independent samples) or from Hi-1- (w + OS Hi-1-; n = 3 biologically independent samples). The GLV composition was determined by SPME-GC-qToF-MS. One-way ANOVA was performed to identify significant differences of E2AL (F2,7 = 32.44, p = 0.0003) or E2OL (F2,7 = 9.887, p = 0.0091) between treatments. Different letters indicate significant differences (p < 0.05) by Tukey HSD post-hoc analysis. Error bars are presented as mean values ± SEM.
