Resting cysts represent a dormant stage in the dinoflagellate life cycle and play crucial roles in bloom-forming species. Once formed, they can settle into marine sediments, thereby prolonging population persistence, or germinate near the sediment surface, contributing to the population dynamics of subsequent generations and sustaining biodiversity over time. However, the physiological and molecular mechanisms that enable resting cysts to survive for extended periods in dark, cold, and oxygen-depleted marine sediments remain poorly understood.Vacuolar H⁺-pyrophosphatase (H⁺-PPase) hydrolyzes inorganic pyrophosphate (PPi) to drive proton translocation across membranes. Compared with the ATP-dependent vacuolar H⁺-ATPase, H⁺-PPase can utilize PPi to maintain proton gradients when ATP availability is limited, suggesting its potential involvement in both cellular pH homeostasis and energy optimization. In this study, we identified and characterized an H⁺-PPase gene, designated SaH⁺-PPase, from the cosmopolitan HAB-forming dinoflagellate Scrippsiella acuminata. Sequence analysis showed that SaH⁺-PPase contains conserved domains characteristic of the H⁺-PPase family and is predicted to localize to the vacuolar membrane, supporting its identity as a typical type I vacuolar H⁺-PPase. To mimic natural sediment conditions, newly formed resting cysts were stored under darkness, 4 °C, and anoxia for 1, 2, and 3 months. Transcriptional analysis showed no significant difference in SaH⁺-PPase expression between vegetative cells and newly formed resting cysts, whereas its expression was markedly upregulated after one month under sediment-mimicking conditions, reaching approximately 2.92-fold that of newly formed cysts, and remained elevated over the three-month incubation period. Parallel reaction monitoring and enzyme activity assays further confirmed that H⁺-PPase protein abundance and activity were significantly increased after three months of incubation compared with those in vegetative cells and newly formed cysts.In natural sediments, resting cysts are frequently exposed to oxygen deprivation, which may lead to ATP deficiency and cytoplasmic acidification. The marked induction of H⁺-PPase at the transcriptional, protein abundance, and enzyme activity levels under dark, cold, and anoxic conditions suggests that H⁺-PPase may contribute to cellular acid-base homeostasis and energy-deficit adaptation through PPi-dependent proton translocation, thereby supporting the long-term survival of resting cysts in sediment environments. These findings provide new molecular insights into the survival mechanisms of dinoflagellate resting cysts and lay a foundation for understanding the physiological adaptation of dinoflagellates in natural benthic habitats.

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