| 摘要: | 研究期間：10108~10207;The plasma membrane plays many vital roles in plant cells, from forming a selective barrier so that a cell can be defined, to providing a suitable environment in which integral proteins can function. Meanwhile, the plant plasma membrane is a direct and major target of heat stress. Maintenance of the integrity of the plasma membrane under heat-stress conditions depends on prompt repair and/or remodeling of the membrane, both of which necessitate the effective transport of membrane components to and from the plasma membrane via directed intracellular trafficking of vesicles. Accumulating evidence has suggested that vesicle tethering, which is mediated by tethering factors, is central to efficient and accurate vesicular trafficking. However, no experimental data have clarified the protective role of tethering factors on the thermostability of the plasma membrane and its effects on heat tolerance in plants. Using a forward genetic approach, we have isolated an EMS-induced hit1-1 (heat-intolerant 1) mutant of Arabidopsis, whose growth is more sensitive to inhibition by high temperature than that of the wild-type. The mutated gene was later identified to encode a subunit of the vesicle tethering complex. Under heat-stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild-type, as determined by an electrolyte leakage assay. Furthermore, hit1-1 was sensitive to the duration (sustained high-temperature stress at 37°C for 3 days) but not the intensity (heat shock at 44°C for 30 min) of exposure to heat, which suggested that HIT1 is involved more in the remodeling than in the repair of plasma membrane. Collectively, HIT1 is the first and so far the only tethering factor linked to thermoadaptation of the plasma membrane. Nevertheless, many fundamental and important questions are still waiting to be answered. For example, is HIT1 responsible for editing plasma membrane by changing lipid composition under heat stress condition? Will this editing alter fatty acid saturation level? Will this alteration affect the fluidity of plasma membrane? Will this affection correlate the thermolabile property observed in hit1-1 plasma membrane? In addition, dose HIT1-mediated vesicle trafficking also influence thermostabillity of other endomembranes such as those of ER, Golgi and vacuole? To answer these critical questions, experiments include fatty acid profiling by gas chromatography, membrane fluidity assay by fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), biogenic membrane thermostability examination by heat-induced chlorophyll fluorescence (changes of Fo in slow heating), and endomembrane thermostability analysis by testing transgenic plants expressing fluorescent proteins (FP) fused with well-established ER, Golgi and tonoplast membrane targeting sequences, will be conducted using both WT and hit1-1 plants grown in either optimal or high temperatures. These results will definitely provide significant information for better elucidating the causal linkage between vesicle trafficking and heat tolerance in plants. In the meantime, characterization of a newly isolated hit4 mutant will be continued. |
