Changes in activities and properties of biosynthetic enzymes in relation to high-temperature-induced proline accumulation in Gracilaria tenuistipitata (Gigartinales, Rhodophyta)

Abstract

The effects of high temperature on proline biosynthesis were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia exposed to 35°C for 4 d. The levels of proline and the specific activities of both Δ1-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) and ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) increased 4 d after exposure to 35°C, during which the apparent Km (P5C) value of P5CR decreased and the apparent Vmax value of δ-OAT increased. Exposure to 35°C did not affect the specific activities of either γ-glutamyl kinase (γ-GK; EC 2.7.2.11) or glutamate-γ-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) but increased their apparent Km and Vmax values. The in vitro application of proline, ranging from 0.01 to 2 mM, inhibited the γ-GK and GSAd of both 25°C and 35°C-treated thalli, with a higher inhibition for 35°C-treated thalli. The δ-OAT and P5CR of both 25°C and 35°C-treated thalli were not affected in the presence of proline. The 35°C-induced increase of proline levels and δ-OAT activity was inhibited by both 6-methylpurine (6-MP, 0.1 mM) and cycloheximide (CHI, 5 μM), but the increase of P5CR activity was inhibited by CHI rather than 6-MP. These observations suggest that at 35°C the glutamate pathway is feedback inhibited by the accumulated proline, and the increase in δ-OAT and P5CR activities is effected at the transcriptional and translational levels. Overall, high temperature induces proline synthesis by a switch of the glutamate pathway to the ornithine pathway via a modification in the activities and properties of biosynthetic enzymes in Gracilaria tenuistipitata.