Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing insects

Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hubner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of wound-responsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.