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Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca 2+ , phosphorylation, and protein-protein interactions on RBOH-dependent ROS production., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Analysis of gene families and identification of homologous genes are important for phylogenetic analysis and for translating results from model to crop species. While numerous plant genomes have been sequenced and made available, the identification of gene models can be difficult, in particular for large gene families arranged in tandem repeats or encoding proteins with a variable number of internal repeats. Thus, correct annotation of plant receptor kinases (PRK) is a challenge. Here, we describe a workflow for the semi-manual extraction, annotation, and verification of genes from annotated gene models as well as from non-annotated DNA regions. This protocol allows the efficient identification of gene family member of PRK from most available plant genomes.