MxRop1-MxrbohD1 interaction mediates ROS signaling in response to iron deficiency in the woody plant Malus xiaojinensis.

Iron (Fe) deficiency affects crop production and quality. Rho of plants (ROPs) involves in multiple physiological processes in plants. While it has not been well characterized under Fe deficiency, especially in perennial woody plants. In our study, we cloned ROP homologous gene MxRop1 from Malus xiaojinenesis, then overexpressed it in Arabidopsis, showing enhanced plant tolerance to Fe deficiency, which demonstrated its gene function during this stress. Overexpression of MxRop1 also increased reactive oxygen species (ROS) levels. Moreover, active state of MxRop1 (CA-MxRop1) interacted with N-terminal region of MxrbohD1, one ROS synthesis gene. When MxrbohD1 was overexpressed in apple calli, it showed significantly increased H2O2 content, fresh weight and FCR activity, while ROS inhibitor application dramatically inhibited FCR activity, demonstrating ROS produced by MxrbohD1 regulated Fe deficiency responses. Furthermore, using Agrobacterium rhizogenes transformation, MxrbohD1 was overexpressed in apple roots, with increased expression of Fe deficiency-induced genes and increased root FCR activity. Under Fe deficiency, it exhibited slight leaf yellowing phenotype. Co-expression of CA-MxRop1 and MxrbohD1 significantly induced ROS generation. Finally, we proposed that MxRop1 interacted with MxrbohD1 to modulate ROS mediated Fe deficiency adaptive responses in Malus xiaojinensis, which will provide a guidance of cultivation of Fe-deficiency tolerant apple plant.