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Global warming has seriously decreased world crop yield. High temperatures affect development, growth and, particularly, reproductive tissues in plants. A gene encoding β-ureidopropionase (SlUPB1, EC 3.5.1.6) was isolated from the stamens of a heat-tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of β-alanine, the only β-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing at 35/30°C (day/night), was 2.16 and 2.93 times greater than that in a heat-sensitive tomato (L4783) cultivated at 30/25°C or 25/20°C, respectively. Transgenic tomatoes, upregulating SlUPB1 in L4783 and downregulating SlUPB1 in CL5915, were constructed, and the amount of β-alanine measured by liquid chromatography-electrospray ionization-mass spectrometry in the transgenic overexpression of SlUPB1 was higher than that of L4783. However, the β-alanine in the transgenics downregulating SlUPB1 was significantly lower than the β-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUPB1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics downregulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of β-alanine further indicated that β-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together, these results showed that the expression of SlUPB1 is important for pollen germination, and β-alanine may play a role in pollen germination under both optimal and high temperatures.