Post‐Translational Modifications Regulate Lipid Remodeling Enzyme During Freezing Stress

As winter approaches, plants acclimate to the cold and begin to induce physiological and metabolic changes that are necessary for survival. While we know much about the signaling pathways that induce metabolic changes at the onset of cold and early freezing, less is known about signaling during severe freezing. One such signaling pathway is the activation of the cryoprotective enzyme Sensitive to Freezing 2 (SFR2). Even though SFR2 is constitutively expressed, it only becomes active as the plant experiences subzero temperatures. The function of SFR2 is to recognize these freezing temperatures and induce membrane remodeling to produce oligogalactolipids, lipids containing two or more galactose molecules as a head group. Because of its ability to sense freezing, many cold‐tolerant species including the model plant Arabidopsis thaliana , require SFR2 activity to survive more severe freezing temperatures. However, the signaling pathway that enables SFR2 to determine when temperatures reach freezing and to become active remains unknown. Here we show that SFR2 activity is regulated by post‐translational modifications, specifically phosphorylations, and that these are the mechanism by which SFR2 can sense a freezing stress. To determine this, we took two complementary approaches. The first used Arabidopsis thaliana exposed to control, cool or freezing conditions. Following temperature treatment, SFR2 was immunoprecipitated and mass spectrometry measurements were taken. From this, we were able to determine a unique phosphorylation pattern on SFR2 at each temperature analyzed. To assess the importance of phosphorylations on the residues of interest we used site‐directed mutagenesis on SFR2 to systematically change the amino acids we found to be phosphorylated to alanine. The effect of these mutations was measured using our second approach, a heterologous expression system in which SFR2 is always active. Functional assays of the SFR2 mutants in this system showed that the residues identified via mass spectrometry were only a portion of those needed for activity. We confirmed this by designing two unique non‐phosphorylatable SFR2s. This was accomplished with in silico modeling of likely phosphorylatable residues, followed by mutagenesis to alanine. Heterologous protein synthesis and function analysis of these non‐phosphorylatable SFR2s suggest these mutants are unable to synthesize oligogalactolipids and are currently being assessed in planta. Using both the Arabidopsis and heterologous expression data we will identify the specific residues required for SFR2 activation and the kinases responsible by determining recognition motifs surrounding the residues. By understanding how metabolic signals change during freezing stress, we can elucidate the dynamics surrounding low‐temperature enzyme activation and direct research for increased cold tolerance in crops.