An assessment of engineered calcium oxalate crystal formation on plant growth and development as a step toward evaluating its use to enhance plant defense

Authors: Nakata, Paul

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/15/2015
Publication Date: 10/30/2015
Citation: Nakata, P.A. 2015. An assessment of engineered calcium oxalate crystal formation on plant growth and development as a step toward evaluating its use to enhance plant defense. PLoS One. 10(10):e0141982.

Interpretive Summary: It has been shown that crystals of calcium oxalate can help protect plants from chewing insects by acting as a physical deterrent making the plant food unpalatable. For humans this may be proportionally similar to having a salad with small gravel like rocks mixed in. This deterring effect coupled with the knowledge that these crystals are largely absent from a number of important crop plants raises the possibility of exploiting this trait for enhancing plant protection. As an initial step toward this realization, this study investigates whether genetically adding these crystals to a non-crystal accumulating plant would cause any deleterious effects to the growth and development of the plant. To conduct this study we first converted the plant A. thaliana from a non-crystal accumulating plant to a crystal accumulating plant by expressing an oxalic acid biosynthetic enzyme within the Arabidopsis plant. By comparing growth and development of the Arabidopsis engineered to make crystals to control plants we were able to determine that adding crystals to non-crystal forming plants does cause them to grow slower and have an altered appearance. These are two negative consequences of adding crystals to plants that do not normally make crystals. In the future, it may be possible to alleviate these negative consequences by altering where in the plant these crystals accumulate, but further work will need to be done before a rational strategy can be designed to engineer crystal formation into non-crystal accumulating plants as a non-pesticide way of protecting plants against chewing insects.

Technical Abstract: The establishment of new approaches to control chewing insects has been sought not only for direct use in reducing crop loss but also in managing resistance to the pesticides already in use. Engineered formation of calcium oxalate crystals is a potential strategy that could be developed to fulfill both these needs. As a step toward this development, this study investigates the effects of transforming a non-calcium oxalate crystal accumulating plant, Arabidopsis thaliana, into a crystal accumulating plant. Calcium oxalate crystal accumulating A. thaliana lines were generated by ectopic expression of a single bacterial gene encoding an oxalic acid biosynthetic enzyme. Biochemical and cellular studies suggested that the engineered A. thaliana lines formed crystals of calcium oxalate in a manner similar to naturally occurring crystal accumulating plants. The amount of calcium oxalate accumulated in leaves also reached levels similar to those measured in the leaves of Medicago truncatula in which the crystals are known to play a defensive role. Visual inspection of the different engineered lines, however, suggested a phenotypic consequence on plant growth and development with higher calcium oxalate concentrations. The restoration of a near wild-type plant phenotype through an enzymatic reduction of tissue oxalate supported this observation. Overall, this study is a first to provide initial insight into the potential consequences of engineering calcium oxalate crystal formation in non-crystal accumulating plants.