Stability and function of rice OsHRZ ubiquitin ligases are regulated at multiple sites by the iron-zinc balance

Iron (Fe) is essential for all living organisms, including plants. Plants upregulate the expression of several genes involved in Fe uptake and translocation in response to Fe deficiency. Hemerythrin motif-containing RING and zinc (Zn)-finger proteins (HRZs) are potential intracellular Fe sensors in plants. Rice (Oryza sativa) OsHRZs are ubiquitin ligases containing hemerythrin domains, a CHY Zn-finger, a CTCHY Zn-finger, a RING Zn-finger, and a rubredoxin-type fold, with each capable of binding Fe or Zn. In this study, we explored whether the OsHRZs sense Fe status by modulating their function depending on metal binding to these domains. The OsHRZ1 protein overexpressed in rice was rapidly degraded in roots, primarily because of the instability of the C-terminal domains. An N-terminal OsHRZ1 sequence with two hemerythrin domains was stably overexpressed in rice and exhibited an enhanced Fe-deficiency response in a dominant-negative manner. CRISPR/Cas9-based rice lines harboring mutations at the C-terminal RING Zn-finger domain or rubredoxin-type fold of OsHRZ1 or OsHRZ2 exhibited Fe-deficiency tolerance and Fe accumulation in grains, indicating the important function of these domains in planta. OsHRZ1 and OsHRZ2 proteins were degraded in vitro through the proteasome pathway, which was dependent on the OsHRZ1 RING Zn-finger domain. Degradation was enhanced under Fe-deficient conditions or with excess Zn bound to the OsHRZ1 protein. The self-ubiquitinating activity of OsHRZ1 and OsHRZ2 was enhanced by excess Zn binding. The results suggest that the stability and function of OsHRZs are affected by Fe nutritional conditions through Zn or Fe binding to multiple domains in the OsHRZ proteins.