Abstract
To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)(1)was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression ofOsPLDα1makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in thepldα1mutant. We also found that the expression ofOsPLDα1is directly regulated by OsDREB1A. This transcriptional regulation ofOsPLDα1could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulatingOsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins.
Highlights
From the ‡Hebei Collaboration Innovation Center for Cell Signaling; Key Laboratory of Molecular and Cellular Biology of Ministry of Education; Hebei Key Laboratory of Molecular and Cellular Biology; College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050024, China; §State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China; ¶State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; College of Biology Science & Engineering, Hebei University of Economics & Business, Shijiazhuang, Hebei 050061, China
Our results suggest that OsPLD␣1 plays a key role in transducing cold signaling in rice by producing phosphatidic acid (PA) and regulating OsDREB1s’ expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins
The CBF/DREB1-dependent cold signaling pathway has been shown to play an important role in regulating cold acclimation in both Arabidopsis and rice
Summary
Protein Extraction and Two-Dimensional Difference Gel Electrophoresis—Because we were interested in finding proteins whose abundance changed rapidly in response to cold stress, a water bath was used for the cold treatment to make sure all the rice seedlings were instantly exposed to a similar temperature at the same time. Nipponbare) rice seedlings were first germinated in double distilled water in darkness for 3 days. Quantitative Real-Time RT-PCR—Seven-day-old wild-type rice seedlings (Dongjin) grown in Hoagland’s liquid growth medium at 28 °C, long-day conditions with 50% humidity were subjected to 12 °C, long-day conditions with 50% humidity for different time intervals. EMSA assay was performed using a modified 10 l mixture, which contained 1.5 l (0.5 g/l) purified protein, 1 l biotin-labeled oligonucleotides, 1 l 10 ϫ binding buffer (100 mM Tris, 500 mM KCl, and 10 mM DTT, pH 7.5), 0.5 l 50% glycerol, 0.5 l 1% Nonidet P-40, 0.5 l 1 M KCl, and 5 l ultrapure water. Rice protoplasts were prepared from leaves of 7-day-old wild-type rice seedlings (Dongjin) plants grown at 28 °C under long-day conditions with 50% humidity. Quantitation of luminescent signal from each of the luciferase reporter enzymes was performed using a dual-luciferase® reporter assay kit (Promega, Fitchburg, WI) according to manufacturer’s instruction
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