Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632-642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2- to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of beta-, gamma-, and delta-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit alpha-tocopherol synthesis. Overexpression of gamma-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of gamma- and delta-tocopherols to alpha- and beta-tocopherols, respectively, indicating that gamma-tocopherol methyltransferase activity limits alpha-tocopherol synthesis in stressed leaves.