Roles for extracellular nucleotides and apyrases in regulating primary root growth in Arabidopsis

When plant cells grow and when they are mechanically stimulated or wounded or attacked by pathogens, they release ATP into their extracellular matrix. This extracellular ATP (eATP) can induce cell signaling changes that alter cell growth and promote adaptive changes to biotic and abiotic stresses. Cells control the eATP concentration with ecto-phosphatases. Among the most important of these are the nucleoside triphosphate-diphosphohydrolases (NTPDases) called apyrases. There are seven apyrases in Arabidopsis, and at least two of these, APY1 and APY2, help control the eATP concentration. The expression of APY1/APY2 can be inhibited by RNAi, and this suppression leads to growth inhibition. Because prior work showed that increased levels of exogenous ATP can block auxin transport and inhibit the gravitropic response in primary roots of Arabidopsis, in this report I tested whether the expression of APY1/APY2 could influence auxin transport and gravitropism. When the expression of these apyrases was suppressed either genetically or chemically, indirect assays of auxin distribution in primary roots showed that that polar auxin transport was interrupted and that, subsequently, the growth and gravitropic curvature of the roots were inhibited. By microarray and qRT-PCR analyses, we assayed how the suppression of APY1/ APY2 by RNAi in R2-4A mutants changed the expression of genes linked to growth inhibition in seedlings. The most significant gene expression changes induced by apyrase suppression were in genes involved in stress responses, which included those regulating wall composition. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and significant changes in reactive oxygen species (ROS) and wall lignification in roots were verified by direct analysis. A group of class III wall peroxidases that are known to be stress response genes and involved in wall modification is up-regulated in the primary roots of R2-4A seedlings when the expression of APY1 and APY2 is suppressed. Those peroxidases use ROS substrates to produce cross-linking and lignification in cell walls. To verify their roles in root growth, a study of peroxidase knockout mutants was performed in the background of R2-4A mutants. When the expression of APY1/APY2 was suppressed in two of these mutants, per54 and per54/49, their root growth was significantly greater than in R2-4A mutants expressing wild type peroxidases. Taken together the results are consistent with the hypothesis that APY1/APY2 and eATP play important roles in the signaling steps that link polar auxin transport to growth, and stress-induced wall changes to growth inhibition.