IC4R004-Proteomic-2007-17849412

Project Title

• Proteomic analysis of rice (Oryza sativa) seeds during germination

The Background of This Project

• Although seed germination is a major subject in plant physiological research, there is still a long way to go to elucidate the mechanism of seed germination. Recently, functional genomic strategies have been applied to study the germination of plant seeds. In this project, the researchers conducted a proteomic analysis of seed germination in rice (Oryza sativa indica cv. 9311) – a model monocot.

Plant Culture & Treatment

• The seeds of the rice (O. sativa cv. 9311) were peeled off and washed with distilled water three times, and then imbibed in distilled water (the water was changed every 24 h) at 267C with 100 mmol photons and m2 s white light in a 12-h light/ dark cycle (12 h day/12 h night). We recorded the seeds’ germination rate, and the changes of 1000 seeds’ weight were recorded during the germination process. Meanwhile, we collected the seeds at 0, 12, 24, 48, and 72 h after imbibition, and they were stored at 2807C until used for protein extraction.

Protein Extraction and 2-D PAGE

• 0.25 g of seeds were ground in 2 mL of precooled homogenization buffer containing 20 mM Tris/HCl (pH 7.5), 250 mM sucrose, 10 mM ethylene glycol-bis(b-aminoethylether)-N,N,N0,N0- tetraacetic acid (EGTA), 1 mM PMSF, 1 mM DTT, and 1% Triton X-100. After homogenization, the homogenate was shifted into a microtube and centrifuged at 15 000 g for 15 min at 47C. The pellet was discarded, and the supernatant was mixed with 1/4 volume 50% cold trichloroacetic acid (TCA) and kept in an ice bath for 30 min. Then it was centrifuged at 15 000 g for 15 min at 47C, and the supernatant was discarded. The pellet was washed with cold acetone three times. After centrifugation, the pellet was vacuum-dried. The dried powder was solubilized in a sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% ampholine (pH 3.5–10), and 20 mM DTT. The protein solution could be used for 2-DE or stored in 2807C until used.
• IEF was carried out using an IPGphor II electrophoresis system and 11-cm immobiline dry strips, with a liner with a pH gradient of 4–7 (GE Healthcare Amersham Biosciences, Little Chalfont, UK). Protein samples (about 900 mg) were loaded during the rehydration step (14 h). IEF was then performed by ramping to 500 V over 1 h, holding at 500 V for 1 h, and 1000 V for 1 h successively, ramping to 8000 V over 1 h, and holding at 8000 V, until a total of 32 kVh were reached. Prior to the second dimension electrophoresis, the gel strips were equilibrated twice as previously described [15]. After equilibration, the strips were applied to vertical SDS-polyacrylamide gels (15% resolving and 5% stacking), and then sealed with 0.5% low-melting agarose in an SDS buffer. Protein markers (GE Healthcare Amersham Biosciences) were loaded beside the strips before sealing. After solidification, electrophoresis was performed in an SDS electrophoresis buffer (pH 8.3), containing 25 mM Tris base, 192 mM glycine and 0.1% SDS, for 3 h at 25 mA. After electrophoresis, gels were stained with CBB R-250. The material preparation and 2-DE were performed for three repetitions to ensure the reliability of the results.
• The stained gels were scanned at a 300 dpi resolution with a UMAX Power Look 2100XL scanner (Maxium Technologies, Taipei, China). The spot detection and gel comparison were made with ImageMaster 2D Platinum, Version 5.01 (GE Healthcare Amersham Biosciences).

Research Findings

• Rice seed weights increased rapidly during the first 20 h imbibition (phase I), and then experienced a stable stage until 50 h (phase II). After these two periods, the seed weights continued to increase (phase III) (Fig. 1).

Figure 1 Changes of the weight for 1000 rice seeds in the germination process.

• 148 protein spots changed in abundance more than two-fold during germination. Among them, 63 decreased, 69 increased (including 20 induced proteins that were absent in the control gels), and another 16 increased first and then decreased (Fig. 2).

Figure 2 2-DE maps of rice seeds in the germination process. Arrows indicate the changed proteins. D, stands for the downregulated protein; M, stands for the complicated changed protein; I, stands for the induced protein which does not exist in the control gels; and U, stands for the up-regulated protein.

• The down-regulated proteins were mainly storage proteins, such as globulin and glutelin, and proteins associated with seed maturation, such as “early embryogenesis protein” and “late embryogenesis abundant protein”, and proteins related to desiccation, such as “abscisic acid-induced protein” and “cold regulated protein”.

• The degradation of storage proteins mainly happened at the late stage of germination phase II (48 h imbibition), while that of seed maturation and desiccation associated proteins occurred at the early stage of phase II (24 h imbibition).

• In addition to a-amylase, the up-regulated proteins were mainly those involved in glycolysis such as UDP-glucose dehydrogenase, fructokinase, phosphoglucomutase, and pyruvate decarboxylase.

Labs working on this Project

• Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,

Chinese Academy of Sciences, Beijing, P. R. China

• College of Life Sciences, Capital Normal University, Beijing, P. R. China
• Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China

Corresponding Author

• shshen@ibcas.ac.cn