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。

A novel rice mutant (Oryza sativa L. var. Zhenhui 249Y) was identified as a low chlorophyll b mutant with a chlorophyll a/b ratio of 4.7. Chloroplast ultrastructure showed decreased grana lamellae and slightly swelling thylakoid in the mutant. Chlorophyll fluorescence assay demonstrated that the mutant noticeably reduced PSII thermostability. However, heat-induced PsbO release from thylakoid membrane indicated an increased thermo-resistance of PSII structure in the mutant. In order to explain these phenomena, two different 2D electrophoresis approaches have been used to compare the thylakoid proteome between the mutant and the wild type: two-dimensional blue native-SDS-PAGE and isoelectric focusing-SDS-PAGE, which was followed by MS identification. Totally, 52 proteins were identified. The results indicated that the LHC-IIb in the mutant was about 40% less than that in wild type. However, both LHC-Ia and LHC-Ib decreased more greatly in this mutant, only 20% remaining. IEF–SDS-PAGE showed that H+-transporting ATP synthase, fructose-bisphosphate aldolase, PSII assembly/stability factor HCF136 increased in the mutant, while PsaE and 8.7 kDa Fe-S protein, decreased. The results suggest that in our mutant, maintenance of the water-oxidizing system is not the major reason of LHC-II stabilization effect on PSII complexes. In addition, the reduction of Chl b affected LHC-I assembly more severely than LHC-II.