Alternative oxidase activity and vacuolar intrusion of mitochondria represent a delayed mitophagy associated with the chilling stress in Haberlea rhodopensis

• Mitochondria intruding into the vacuole appear in H. rhodopensis under chilling. • Increased AOX activity co-occur with vacuole intruded mitochondria. • Mitophagy is only completed after subjected to sub-zero temperatures. • Existence of mitochondria intruded into the vacuole represents a delayed mitophagy. • Delayed mitophagy is suggested as a chilling stress tolerance mechanism. The resurrection plant Haberlea rhodopensis exhibits tolerance to both desiccation and sub-zero temperatures. While the overlap between the tolerance to desiccation and sub-zero temperatures has been previously described, specific responses to above-zero chilling stress remained poorly understood. Mitochondrial alternative oxidase (AOX) activity is associated with chilling stress tolerance, particularly in plants of (sub)tropical origin, the function of which has not been well resolved in the chilling stress tolerance of resurrection plants. In H. rhodopensis leaves naturally exposed to chilling conditions, transcript abundance, protein level, and respiration activity of AOX significantly increased. Ultrastructural analysis revealed that chilling conditions resulted in the intrusion of the vast majority of mitochondria into the central vacuole, where mitochondria were represented in multiple cross sections resembling to immersion heaters. Ultrastructure analysis revealed that mitochondria intruding into the vacuole undergo a gradual degradation process thus considered as mitophagy. In consequence in leaves naturally exposed to sub-zero conditions, the degradation of the vacuole intruding mitochondria is nearly complete associated with a decreased AOX activity and protein amount. Leaf temperature alterations under chilling conditions and after sub-zero conditions support the heat generation based on the increased AOX activity under chilling conditions. Our findings revealed an adaptive strategy in H. rhodopensis to chilling conditions, where coordinated AOX-driven respiration and delayed mitophagy, enabling a temporal existence of mitochondria intruded into the vacuole together support metabolic stability.