| Summary: | <i>Brassica napus</i> L. is a vital plant oil resource worldwide. The fatty acid biosynthesis and oil accumulation in its seeds are controlled by several genetic and environmental factors, including daytime and nighttime temperatures. We analyzed changes in oleic and erucic acid content in two double haploid (DH) lines, DH0729, a weakly temperature-sensitive line, and DH0815, a strongly temperature-sensitive line, derived from <i>B. napus</i> plants grown at different altitudes (1600, 1800, 2000, 2200, and 2400 m a.s.l., 28.85° N, 112.35° E) and nighttime temperatures (20/18, 20/16, 20/13 and 20/10 °C, daytime/nighttime temperature). Based on medium- and long-chain fatty acid metabolites, the total oleic acid content 35 and 43 days after flowering was significantly lower in low nighttime temperature (LNT, 20/13 °C) plants than in high nighttime temperature (HNT, 20/18 °C) plants (HNT: 58–62%; LNT: 49–54%; an average decrease of 9%), and the total erucic acid content was significantly lower in HNT than in LNT plants (HNT: 1–2%; LNT: 8–13%; an average increase of 10%). An RNA-seq analysis showed that the expression levels of <i>SAD</i> (LOC106366808), <i>ECR</i> (LOC106396280), <i>KCS</i> (LOC106419344), <i>KAR</i> (LOC106367337), <i>HB1</i>(LOC106430193), and <i>DOF5</i> (LOC111211868) in STSL seeds increased under LNT conditions. In STSL seeds, a base mutation in the <i>cis</i>-acting element involved in low-temperature responsiveness (LTR), the <i>HB1</i> and <i>KCS</i> promoter caused loss of sensitivity to low temperatures, whereas that of the <i>KCS</i> promoter caused increased sensitivity to low temperatures.
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