Rho of plant GTPase MxROP1 regulates the responses to Fe deficiency by targeting Zinc Ribbon 3 in apple rootstocks

Abstract

The Rho-related GTPase of plants (ROPs) is a multifunctional molecular switch that can regulate plant growth, development, and responses to the environment. However, how the ROPs regulate the response to Fe deficiency has not been thoroughly clarified. In this study, we found that Fe deficiency induced MxROP1 in apple (Malus xiaojinensis) at both the transcriptional and translational levels. The overexpression of MxROP1, MxROP1DN (inactive form), and MxROP1CA (active form) in apple roots increased the activity of ferric chelate reductase and the ability to acidify the rhizosphere, and the lines that overexpressed MxROP1DN exhibited the strongest reaction to enhanced Fe uptake. Yeast two-hybrid library screening indicated that MxROP1 interacted with MxZR3.1, a DNL zinc finger protein that negatively regulates the responses to Fe deficiency. We identified their interaction in vitro and in vivo in more detail using pull-down and bimolecular fluorescence complementation assays, respectively, and MxROP1DN-MxZR3.1 interacted the most strongly. Furthermore, MxROP1 negatively affected the stability of MxZR3.1 protein in vitro as shown by a cell semi-degradation assay, and the application of MG132 inhibited the degradation of MxZR3.1-HIS proteins. This indicated that MxROP1 caused the degradation of MxZR3.1 protein through the 26 S proteasome pathway. Similar results were found in OE-MxROP1+OE-MxZR3.1 transgenic apple calli compared with those in the OE-MxZR3.1 calli. We also demonstrated that MxZR3.1 interacted with MxbHLH39, a known positive transcription factor and core component of Fe deficiency, and MxROP1 affected the interaction of MxZR3.1-MxbHLH39 as shown in a competitive binding assay. Τhis clarified one MxROP1-MxZR3.1-MxbHLH39 pathway that maintains Fe homeostasis in M. xiaojinensis.