Haberlea rhodopensis is a homoiochlorophyllous resurrection plant with remarkable
tolerance to both
desiccation and sub-zero temperatures. While the overlap between drought and freezing
tolerance
has been previously established, specific mitochondrial responses to chilling stress
remained poorly
understood. This study elucidates the chilling tolerance mechanisms of H. rhodopensis,
focusing
on mitochondrial behaviour and alternative oxidase (AOX)-mediated respiration. Under
chilling
conditions (above-freezing low temperatures), HrAOX2 transcript abundance, AOX protein
levels
and AOX-dependent respiration significantly increased—up to four-fold—compared to
control
plants. This shift in mitochondrial activity occurred alongside localized warming
in the leaves, as
detected by thermal imaging, suggesting that AOX activity contributes to cellular
heat generation
even in non-thermogenic plant tissues. Crucially, ultrastructural analysis revealed
the unusual
relocation of mitochondria into the central vacuole, where they appeared to undergo
a slow and
spatially controlled degradation process. This "delayed mitophagy" likely enables
mitochondria to
maintain AOX activity and contribute to thermogenesis and redox homeostasis before
their eventual
breakdown. The vacuolar sequestration of mitochondria may also protect the cytoplasm
from
excessive reactive oxygen species production during stress. Upon prolonged exposure
or sub-zero
temperatures, these mitochondria disintegrated, correlating with reduced AOX activity
and loss of
thermal buffering capacity.
Our findings reveal a sophisticated, multi-level adaptive strategy in H. rhodopensis,
where coordinated
AOX-driven respiration and regulated mitophagy support metabolic stability and thermal
protection
during chilling stress. This mechanism likely co-evolved with desiccation tolerance
and may inform
future approaches for improving cold resilience in crop species under climate change.
Keywords: Haberlea rhodopensis; mitophagy; alternative oxidase; thermogenesis, chilling
stress
Acknowledgements: This work was supported by the grant K-146865 of NKFIH, Hungary,
and by
the bilateral mobility grant between the Hungarian and the Bulgarian Academies of
Sciences. Á.S.
was supported by the János Bolyai Scholarship of the Hungarian Academy of Sciences
under grant
number BO-00113-23-8.
