Glycosylated Sterols Enhance Cold Tolerance in Tomato via Membrane Stabilization and Jasmonate Signaling

Free and glycosylated sterols play a central role in maintaining the structural integrity and proper functioning of plasma membrane, which serves as the primary sensor of cold and initiates signaling cascades to mitigate chilling-induced damage. Here we characterize the cold response of tomato (Micro-Tom) mutants with opposite glycosylated to free sterol ratio resulting from the overexpression and silencing of the sterol glycosyltransferases SlSGT2 and SlSGT1. A high ratio in the SlSGT2 overexpressing mutants increases cold tolerance, membrane stability, and oxidative stress responses, while a low ratio in the SlSGT1-silenced mutants causes the opposite phenotypes. The different cold sensitivity in these mutants is due to the altered membrane sterol profiles, rather than to a different capacity to adjust sterol levels during cold exposure. Changes in the glycosylated to free sterol ratio also trigger distinct transcriptional programs that establish a preconditioned stress-responsive state under basal conditions and a more efficient response under cold exposure in the SlSGT2 overexpressing mutants and compromise the capacity to withstand the effects of cold stress in the SlSGT1-silenced mutants. The SlSGT2 overexpressing mutants accumulate higher basal levels of jasmonates and display enhanced biosynthetic capacity under cold stress compared to SlSGT1-silenced and control plants. The elevated glycosylated to free sterol ratio in SlSGT2 overexpressing mutants facilitates JA biosynthesis and signaling, leading to the activation of cold-responsive genes, including those of the CBF–COR pathway, antioxidant defenses and polyamine biosynthesis. Our findings provide key insights into the mechanisms by which glycosylated sterols improve cold tolerance in tomato. Overall design: Tomato (S. lycopersicum cv. Micro-Tom) plants were grown in pots containing a substrate mixture of peat (Klasmann TS2), perlite, and vermiculite (3:1:1) under controlled environmental conditions. The growth chamber was set to a 16-h photoperiod (light intensity: 200 µmol m?² s?¹) and an 8-h dark period, with a day/night temperature of 25/22°C. When plants were one-month-old, half of the them were transferred to a growth chamber maintained at 4°C while retaining the same light/dark regime, whereas the remaining plants continued under control conditions. Leaf samples (third and fourth leaves) were collected at different time points from both control and cold-treated plants, and either immediately analyzed or frozen in liquid nitrogen and stored at -80°C for further processing.