1. Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK
2. Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
3. Marine Biological Association, Citadel Hill, Plymouth PL1 2PB, UK
4. Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK
5. These authors contributed equally to this work
6. Present address: Department of Biology and Curriculum in Genetics and Molecular Biology, CB3280, 108 Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280, USA.

Correspondence to: Liam Dolan¹ Correspondence and requests for materials should be addressed to L.D. (e-mail: Email: liam.dolan@bbsrc.ac.uk).

Abstract

Cell expansion is a central process in plant morphogenesis, and the elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca²⁺ influx from the extracellular store is required for (and sets the rates of) cell elongation in roots¹. Arabidopsis thaliana rhd2 mutants are defective in Ca²⁺ uptake and consequently cell expansion is compromised—rhd2 mutants have short root hairs^{2, 3} and stunted roots. To determine the regulation of Ca²⁺ acquisition in growing root cells we show here that RHD2 is an NADPH oxidase, a protein that transfers electrons from NADPH to an electron acceptor leading to the formation of reactive oxygen species (ROS). We show that ROS accumulate in growing wild-type (WT) root hairs but their levels are markedly decreased in rhd2 mutants. Blocking the activity of the NADPH oxidase with diphenylene iodonium (DPI) inhibits ROS formation and phenocopies Rhd2^-. Treatment of rhd2 roots with ROS partly suppresses the mutant phenotype and stimulates the activity of plasma membrane hyperpolarization-activated Ca²⁺ channels, the predominant root Ca²⁺ acquisition system. This indicates that NADPH oxidases control development by making ROS that regulate plant cell expansion through the activation of Ca²⁺ channels.