Unstructured regions differentially modulate the activation of RBOHD and RBOHH

Abstract

Reactive oxygen species (ROS) produced by plant NADPH oxidases (RBOHs) must be precisely controlled to generate appropriate signaling outputs in profoundly diverse physiological contexts. RBOHs are activated by Ca2+; and phosphorylation, yet how structural variation in their regulatory regions shapes these responses and contributes to functional specialization remains unclear. To identify regions underlying functional diversification among RBOHs, we performed phylogenetic and sequence-comparison analyses across land plants. The most variable regions are two N-terminal unstructured regions (URs), UR1 and UR2, which flank the EF-hand Ca2+;-binding domain (EFD). We investigated whether these unstructured regions contribute differently to the regulation of Arabidopsis RBOHD and RBOHH, which function predominantly in immune signaling and pollen tube growth, respectively. Our functional assays in HEK293T cells suggest that UR1 plays opposing roles in RBOHD and RBOHH, restraining Ca2+;-dependent activation in RBOHD while facilitating it in RBOHH. Our analyses indicate differences in the regulatory contribution of UR2 between RBOHD and RBOHH. In RBOHD, our results suggest that UR2 phosphorylation promotes formation of an -helical conformation and its interaction with the catalytic domain, contributing to enzymatic activation, whereas our analyses do not support a comparable regulatory mechanism in RBOHH. Domain-swapping experiments between RBOHD and RBOHH suggest that the EFD coevolved with UR2 in RBOHD to enhance phosphorylation-dependent activation, whereas in RBOHH it coevolved with UR1 to favor Ca2+;-dependent activation. Together, our findings support a model in which diversification of unstructured regulatory regions adapts the conserved RBOH enzymatic core to distinct physiological contexts, including immune signaling and polarized growth.