Cytosolic Ca2+ mediates ATG8i-autophagy activation under osmotic stress in Arabidopsis thaliana

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Osmotic stress–induced Ca 2 ⁺ accumulation promotes the formation of specific RFP-ATG8i labeled autophagosome near the hypocotyl-root transition zone, suggesting ATG8i-dependent autophagy or reticulophagy, as a mechanism to alleviate osmotic and endoplasmic reticulum stress in plants. Autophagy is a crucial and evolutionarily conserved process that breaks down and recycles damaged or unnecessary cytoplasmic components. This process is essential for plant growth and for responding to environmental stresses. In this study, we investigated how autophagy is regulated during osmotic stress in Arabidopsis thaliana , with a focus on the behavior of the RFP-tagged ATG8i protein and the potential role of cytosolic Ca 2 ⁺ in this process. Osmotic stress was induced with sorbitol and Ca 2+ accumulation was monitored with a Cameleon transgenic line. Ca 2 ⁺ influx from the apoplast was disrupted using EGTA, and autophagy was analyzed in a transgenic line expressing RFP-ATG8i under its native promoter. We found that osmotic stress triggers Ca 2 ⁺ accumulation, with a pronounced response in the hypocotyl-root transition zone and a weaker response at the root tip. Both osmotic stress and Ca 2 ⁺ signaling promote the accumulation of RFP-ATG8i-labeled autophagosomes in predominantly in hypocotyl-root transition zone. Furthermore, analysis of GFP-HDEL/RFP-ATG8i doubled transgenic line revealed colocalization of RFP-ATG8i with the endoplasmic reticulum marker HDEL, suggesting that ATG8i participates in reticulophagy and may contributed to the ER turnover under stress conditions.