Multi-level evidence reveals a possibly vital role of H+-pyrophosphatase in the energetic metabolism of dinoflagellate resting cysts.

As a dormant stage of the dinoflagellate life cycle, resting cysts play crucial ecological roles, particularly for bloom-forming species, as supported by abundant evidence. They can remain viable in marine sediments for decades to centuries and directly regulate seasonal population dynamics and initiate HABs through germination. Yet the physiological and molecular mechanisms that enable their long-term survival under dark, cold, and anoxic conditions remain largely elusive. Vacuolar H⁺-pyrophosphatase (H⁺-PPase) is a conserved proton pump that hydrolyzes inorganic pyrophosphate (PPi) to drive proton translocation, thereby conferring tolerance to anoxia and cold in plants. Here, we identified and characterized an H⁺-PPase gene (SaH⁺-PPase) from the cosmopolitan bloom-forming dinoflagellate Scrippsiella acuminata. Sequence analysis confirmed that SaH⁺-PPase is a typical type I vacuolar H⁺-PPase with conserved domains and vacuolar membrane localization. Transcriptional profiling revealed significant induction of SaH+-PPase in resting cysts under sediment-mimicking conditions (darkness, 4 °C, anoxia), with the expression elevated ∼2.9-fold after one month and sustained for three months. Consistently, parallel reaction monitoring (PRM) and enzyme activity assays verified sustained increases in H⁺-PPase protein abundance and activity during three months of incubation. Since oxygen deprivation sharply reduces cellular ATP levels and triggers cytoplasmic acidification, the markedly upregulation of H⁺-PPase likely not only helped maintain cellular homeostasis by alleviating cytoplasmic acidosis but also enabled resting cysts to utilize PPi as an alternative energy source to compensate for energy deficits under dark, cold, and anoxic conditions, thereby directing limited ATP toward essential processes for long-term dormancy. Collectively, our findings provide novel molecular insights into the survival mechanisms of resting cysts and lay a foundation for understanding the physiological adaptations of dinoflagellates in natural benthic habitats.