Antioxidant enzymes and the mitochondrial alternative oxidase pathway play important roles in chilling tolerance of Haematococcus pluvialis at the green motile stage

Abstract During outdoor large-scale cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to chilling stress in winter. However, the physiological responses and adaptation mechanisms to chilling in H. pluvialis have not been characterized. In order to better understand the chilling tolerance mechanisms of H. pluvialis in the green motile stage, the responses of photosynthetic characteristics and photoprotective mechanisms to chilling were investigated. Chilling stress significantly decreased the activities of key enzymes (ribulose-1,5-bisphosphate carboxylase/oxygenase and NADP glyceraldehyde-3-phosphate dehydrogenase) in photosynthetic carbon assimilation, which would cause the accumulation of excess reducing equivalents and an imbalance of light absorption and energy utilization, leading to more severe photoinhibition. After chilling treatment, there was no induction of cyclic electron flow, non-photochemical quenching, fluorescence emission, or catalase, indicating these factors do not protect H. pluvialis from photoinhibition. However, chilling significantly enhanced the activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase), which efficiently dissipated excess electrons generated by photosynthetic linear electron flow (LEF). The malate-oxaloacetate shuttle was activated and the mitochondrial alternative oxidase (AOX) pathway was significantly up-regulated after chilling, indicating the transport of excess reducing equivalents generated by photosynthetic LEF to cytosol and mitochondria and oxidization by the AOX pathway, the mitochondrial non-phosphorylating pathway. As a result, the LEF was not inhibited by negative feedback and the H. pluvialis cells were well protected under chilling stress in the light, with no change in the effective quantum yield of PSII (ΦPSII) and the yield of non-regulated energy dissipation of PSII (ΦNO). The results indicate that antioxidant enzymes and the mitochondrial AOX pathway play important roles in protecting H. pluvialis at the green motile stage against chilling stress.