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Cotton is an important agricultural crop to many regions across the globe but is sensitive to low-temperature exposure. The activity of the enzyme SENSITIVE TO FREEZING 2 (SFR2) improves cold tolerance of plants and produces trigalactosylsyldiacylglycerol (TGDG), but its role in cold sensitive plants, such as cotton remains unknown. Recently, it was reported that cotton SFR2 produced very little TGDG under normal and cold conditions. Here, we investigate cotton SFR2 activation and TGDG production. Using multiple approaches in the native system and transformation into Arabidopsis thaliana, as well as heterologous yeast expression, we provide evidence that cotton SFR2 activates differently than previously found among other plant species. We conclude with the hypothesis that SFR2 in cotton is not activated in a similar manner regarding acidification or freezing like Arabidopsis and that other regions of SFR2 protein are critical for activation of the enzyme than previously reported.