Bitterness in citrus fruit extract is caused by changes and transformations in limonoids and the production of limonene, a bitter-tasting substance. Limonene synthase enzyme is responsible for producing limonene. This study examined the effect of low-temperature stress on the expression of the limonene synthase gene in four different citrus species: Miyagawa mandarin (Citrus unshiu), Thomson Navel Orange (Citrus sinensis var. Thomson navel), Ruby Star grapefruit (Citrus paradisi var. Ruby star), and Lisbon lemon (Citrus limon var. Lisbon). These species were grafted on two-year-old sour orange rootstocks. The experiment was carried out as a factorial in the form of a completely random design. Three temperatures were used for the cold treatment (−8, −4, 4 °C). The expression sequence of the Limonene synthase gene was predicted using bioinformatic software such as PLANT Care and PlantPan. The results showed significant variation in the frequency of amino acids in the LS gene. Leucine (11.36) and glutamic acid (7.57) had the highest frequency. The analysis of the Limonene synthase gene promoter revealed different Cis-element regions such as response elements to light, hormones, and abiotic stresses on the promoter of these genes. The gene promoter had many regions related to binding sites of MYB transcription factors, response regions to hormones, cold (Dehydrin with 15 cis-regulatory elements), and drought, emphasizing the role of this gene in abiotic stress response. The gene expression patterns of the Limonene synthase showed that the highest and lowest expression occurred at − 4 °C in Lisbon lemon (−132.28), Ruby Star grapefruit (−70.06), Thomson Navel Orange (−2.88), and Miyagawa Mandarin (−3.88), confirming the role of this gene in abiotic and biotic stress response.
