HIGD2 confers hypoxic stress tolerance in rice by improving mitochondrial function

Flooding and oxygen deprivation severely impair mitochondrial energy metabolism in rice, reducing growth and survival under waterlogged conditions. To elucidate the mechanisms of mitochondrial adaptation during hypoxia, we characterized OsHIGD2, a rice gene encoding a hypoxia-induced gene domain (HIGD) protein. Transgenic rice lines overexpressing OsHIGD2 and CRISPR/Cas9 knockout mutants were generated to investigate its functional role under low-oxygen stress. The expression of OsHIGD2 was strongly induced by submergence and hypoxia, particularly during the early stress phase. Overexpression lines exhibited markedly improved survival and recovery after 7 days of hypoxic treatment, showing two-to threefold increases in leaf number, length, and area compared with wild-type controls. Conversely, knockout mutants displayed growth retardation and enhanced electrolyte leakage, indicating greater cellular damage. Overexpression lines maintained significantly higher cytochrome c oxidase (complex IV) activity under hypoxia, whereas mutants showed decreased activity, suggesting that OsHIGD2 contributes to sustaining mitochondrial respiration during oxygen limitation. No significant differences in NAD⁺ content were observed among the lines, implying that OsHIGD2-mediated tolerance mainly involves preservation of mitochondrial integrity rather than glycolytic adjustment. Collectively, these results demonstrate that OsHIGD2 enhances hypoxia tolerance in rice by maintaining mitochondrial function and respiratory efficiency, offering insights into mitochondrial regulation of flood resilience in crops.