CRISPR/Cas9-Engineered Large Fragment Deletion Mutations in Arabidopsis CEP Peptide-Encoding Genes Reveal Their Role in Primary and Lateral Root Formation.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are post-translationally modified peptides that play essential roles in root and shoot development, nitrogen absorption, nodule formation and stress resilience. However, it has proven challenging to determine biological activities of CEPs because of difficulties in obtaining loss-of-function mutants for these small genes. To overcome this challenge, we thus assembled a collection of easily detectable large fragment deletion mutants of Arabidopsis CEP genes through the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9-engineered genome editing. This collection was then evaluated for the usability by functionally analyzing the Arabidopsis growth and development with a focus on the root. Most cep mutants displayed developmental defects in primary and lateral roots showing an increased primary root length and an enhanced lateral root number, demonstrating that the genetic resource provides a useful tool for further investigations into the roles of CEPs.

Superoxide anion generation response to wound in Arabidopsis hypocotyl cutting.

Cutting is a frequently used model to study the process of adventitious root formation, and excision of cuttings leads to rapid wound response signaling. We recently showed that as a wound signal, reactive oxygen species (ROS, mainly hydrogen peroxide) participate in adventitious root induction of hypocotyl cuttings through regulation of auxin biosynthesis and transport. Here, superoxide anion (O2-•), an early type of ROS, exhibited rapid burst at the cutting site immediately in response to wounding in Arabidopsis hypocotyl cuttings. Diphenylene iodonium chloride (DPI, inhibitor of NADPH oxidase) overwhelmingly suppressed O2-• propagation through the hypocotyl. Compared to wild type, O2-• burst only occur in cut base, and upward transduction were inhibited completely in NADPH oxidase mutant AtRbohD. These results indicate O2-• generation and propagation in response to wound and via NADPH oxidase in adventitious root induction of hypocotyl cuttings.