The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 f Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H+ symport, Arrhenius plots with wild-ype permease exhibit a discontinuity at 18-19℃ with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H+ symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325f Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H+ translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H+ translocation rather than the conformational change (s) responsible for translocation across the membrane.

To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma-membrane-associated proteins following treatment with 150mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt-sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two-phase partitioning, and plasma-membrane-associated proteins were separated by IEF/SDS-PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5-fold in response to salt stress. Of these proteins, nine were upregulated and nine were down-regulated. MS analysis indicated that most of these membraneassociated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine-rich-repeat type receptor-like protein kinase, OsRPK1, was identified as a salt-responding protein. Immuno-blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno-histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.

It has been shown that mitochondria play a pivotal role in plant programmed cell death (PCD). Previous study established a salt stress-induced PCD model in rice (Oryza sativa L. cv. WYJ 8th) root tip cells, demonstrated by DNA laddering, cytochrome c release, and TUNEL positive reaction. In this study, the role of mitochondria during the early phase of PCD (2h-PCD) was analyzed in rice roots. After 2h-PCD induction, the integrity of mitochondria decreased slightly, consistent with a small release of cytochrome c. 2h-PCD partially inhibited electron transport, resulting in oxidative burst in mitochondria. However, ATP production maintained constant. Mitochondria proteome were analyzed by two-dimensional IEF/SDS-PAGE before and after 2h-PCD induction, and eight PCD-related proteins were identified. Among them, four proteins were up-regulated after PCD induction, which included glycoside hydrolase, mitochondrial heat shock protein 70, 20S proteasome subunit, and Cu/Zn-superoxide dismutase, and four were down-regulated, namely ATP synthase beta subunit, cytochrome c oxidase subunit 6b, S-adenosylmethionine synthetase 2, and transcription initiation factor eIF-3 epsilon. These results suggested that ATP synthase may not be the major producer of ATP in mitochondria during the early stage of PCD in rice. Glycoside hydrolase may be involved in ETC impairment and ROS burst, and mitochondrial HSP70 is a potential candidate for PCD regulation. The possible roles of other proteins on PCD initiation were also discussed.

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20 kDa (OsCyt20) and a nuclear of 37 kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca2+/Mg2+-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn2+-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4 h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.

前期研究表明，水稻根尖细胞质膜类受体蛋白激酶OsRLK的表达受盐胁迫诱导。为了进一步研究该激酶的生理功能，通过反转录PCR得到OsRLK胞外区cDNA片段，将其亚克隆至pET29a原核表达载体并在大肠杆菌中实现了高表达，表达量约为细胞总蛋白的30%。重组蛋白经SDS-PAGE分离，染色切胶收集后，作为抗原免疫新西兰家兔，分离抗血清，经纯化得到1：20000效价的多克隆抗体。Western blot结果显示，该抗体能特异识别在原核表达系统内表达的抗原，以及水稻根尖细胞质膜组分中的LRR型类受体蛋白激酶，并且在蛋白质水平证实该激酶为盐胁迫响应蛋白。