Hormone analysis
Jasmonates being the important signalling hormones induced in plants after caterpillar herbivory, free JA, amino-acid conjugate of JA (JA-Ile), and OPDA were measured in wild type and an ethylene insensitive skl mutant of M. truncatula. Concentration of free JA as well as JA-Ile was significantly higher in plants fed by caterpillars with or without intact spinneret than control in both genotypes. But the level of OPDA was variable across treatments and not significantly different in caterpillar damaged plants from control. It is observed that higher level of free JA and JA-Ile is accumulated in wild type plants than in skl mutants after caterpillar herbivory which indicates the effect of ethylene is JA biosynthesis or JA signalling pathway.
ACC oxidase is an enzyme that catalyzes the last step in ethylene biosynthesis pathway (Xu et al., 2008). It oxidizes ACC to liberate ethylene. In a feed forward mechanism, ACC oxidase is also responsive to hormone ethylene (Alexander and Grierson, 2002). Thus the relative transcript level of ACC oxidase was compared between control and caterpillar infested plants (both with and without intact labial salivary gland) as an marker of both ethylene production and ethylene responsive gene. But no significant difference in the transcript level due to herbivory was seen in both genotypes which showed that the production of ethylene is not affected by herbivory. However, it cannot be ruled out that the level of hormone might have levelled off at the time of sample collection which was done after 10 hours of herbivory.