Katharina Ebel

• Although ABCG7 protein levels decrease in the plastid proteome under cold conditions (Trentmann et al., 2020), the physiological role of the plastid ABC-type transporter ABCG7 during cold acclimation and freezing stress remains poorly understood and its localisation had not been experimentally confirmed. Employing transient expression of ABCG7-GFP in Nicotiana benthamiana and proteolytic assays in isolated chloroplasts, ABCG7 was shown to localise to the outer chloroplast envelope. Characterisation of abcg7 loss-of-function mutants under cold stress revealed increased biomass accumulation, decreased glucose and starch levels, and elevated Krebs cycle intermediates. Metabolic changes were accompanied by increased anthocyanin synthesis and decreased phenylalanine levels, suggesting a shift of phenylalanine flux towards secondary metabolism, potentially contributing to enhanced cold tolerance. Further, a striking galactose accumulation was observed in abcg7 mutants during cold acclimation. When seedlings were grown on galactose supplemented plates, anthocyanin synthesis in abcg7 mutants was observed, and may explain the observed phenylalanine depletion. Despite the cold tolerance, abcg7 mutants exhibited increased sensitivity to freezing. Expression and cell wall sugar analyses during post-freezing stress revealed altered cell wall composition, indicating cell wall fragility under sub-zero conditions. Freezing stress also triggered increased ABCG7 expression in wild types, suggesting a specific role in post-freezing recovery Figure 18C. Given that plastid membranes are rich in galactolipids and highly vulnerable to freezing injury, the lipid composition and expression of membrane remodelling genes were analysed. In abcg7 mutants, lower levels of phosphor-, galacto- and storage lipids were detected during thawing, alongside with an altered expression pattern of the membrane remodelling enzyme SFR2. BiFC analysis confirmed a physical interaction between ABCG7 and SFR2, indicating a possible role for ABCG7 in the regulation of SFR2 activity and plastid membrane remodelling during freezing stress.