IC4R003-Proteomic-2016-16548068

Project Title

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth

The Background of This Project

• In flowering plants, the highly specialized haploid male plant (pollen or male gametophyte) generated from diploid micro- sporocytes in anthers of stamen is a key regulator of sexual reproduction and contributes to selection of vigorous off-spring and genetic diversity of population.

In this study, the researchers report for the first time, their analysis of the protein complement of mature pollen using 2-DE with MALDI-TOF MS and ESI Q-TOF MS/MS, and 1-D SDS PAGE with nano-LC ESI Q-TOF MS/MS. These analyses identified 322 unique proteins, most of which have not been reported previously to be in pollen.

Plant Culture & Treatment

• Rice cultivar Zhonghua 10 (Oryza sativa L. ssp japonica) was grown under natural conditions. The plants were fertilized and flooded normally. Mature pollen grains were collected by shaking panicles gently during flowering, and were used or stored at 2807C immediately.

Figure 1. Cytological observation of fresh mature pollen and eluted mature pollen.

Protein Extraction and 2-D PAGE

• To prepare pollen-released proteins (PRPs), 0.5 g of pollen grains were suspended in 5 mL elution buffer (0.7 M sucrose, 0.5 M Tris-HCl, pH 7.2, 50 mM EDTA, 10 mM KCl, 2 mM PMSF, 13 mM DTT) and incubated with gentle shaking on ice for 15 min. Eluate (containing PRPs) was collected by centrifuged at 506g for 10 min, and the pelleted pollen grains were used in the next cycle of elution. This procedure was repeated until the proteins in the eluate were hardly detected by the Bradford method. After each elution, the intactness of the eluted pollen grains was checked under microscope. The eluates were combined and then centrifuged at 18 0006g for 20 min.

Figure 2. Protein concentrations in each eluate (A) and the I 2 -KI reaction of pollen (B and C, bar = 80 mm) during PRP preparation.

• The resultant supernatant was used as the PRP fraction. The PRPs were precipitated with 12.5% tri- chloroacetic acid on ice for 2 h, and then collected by centrifugation at 15 0006g for 20 min at 47C. The pelleted PRPs were resuspended in 80% cold acetone containing 0.07% b- mercaptoethanol, cooled at 2207C for 30 min, and finally collected by centrifuged at 15 0006g for 20 min at 47C. After being rinsed with cold acetone with 0.07% b-mercaptoethanol and dried by vacuum, the resultant proteins were dissolved in protein lysis buffer (7 M urea, 2 M thiourea, 4% v/v NP-40, 13 mM DTT, 2% v/v pharmalyte 3–10) and used for 2-DE immediately or stored at 2807C after debris was removed by centrifugation at 20 0006g for 20 min at 47C.
• The eluted pollen grains, used to extract pollen-interior proteins (PIPs), were homogenized in a homogenate buffer (100 mM Tris-HCl pH 7.6, 5 mM KCl, 2% SDS, 2% NP-40, 1% b-mercaptoethanol) with chilled mortar and pestle. After almost all pollen grains were broken (confirmed by micros- copy), the homogenate was centrifuged at 18 0006g for 20 min and the resultant supernatant was used for prepara- tion of PIPs by the same procedure described above. Protein concentrations were determined according to the Bradford method [13] by DU640 UV-visible spectrophotometry (Beckman). BSA was used as the standard.
• To collect pollen coat-associated proteins, the freshly collected mature pollen grains (100 mg) were eluted by 500 mL diethyl ether for about 1 min, and centrifuged at 500 6g. The collected supernatant was vaporized by SpeedVec (Ther- moSavant), resolved by SDS sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromo- phenol blue), and then subjected to standard 1-DE with 12.5% SDS polyacrylamide gel. The resulting gel was briefly stained by CBB G250, and the detected protein bands were cut into horizontal slices (14 in total).
• An aliquot (about 600 mg proteins) of PRP or PIP sample was diluted with rehydration buffer (7 M urea, 2 M thiourea, 1% v/v NP-40, 13 mM DTT, 0.5% IPG Buffer 3–10, 0.002% Bromophenol Blue), loaded onto an IPG strip holder with 24 cm, pH 3–10 or pH 4–7 linear gradient IPG strips (Amersham Biosciences, Sweden) and was run in an Ettan IPGphor Isoelectric Focusing System following the protocol of the manufacturer. For SDS-PAGE, the equilibrated IPG gel strips were placed onto 12.5% ExcelGel SDS gels (Amersham Pharmacia Biotech) using an Ettan DALT Six Electrophoresis Unit. Low molecular-mass (MM) protein markers (Fermentas, Canada) were co-electrophoresed and used as MM standards. The proteins in gels were visualized by CBB staining. The experiments were repeated 3 times for each preparation of PRPs and PIPs.

Research Findings

• The researchers characterized the microstructure and ultrastructure of maturerice pollen under light microscopy, SEM and TEM. Maturerice pollen is a tricellular organism (Fig. 1A) and has abundant mitochondria, endoplasmic reticulum and vesicles (Fig. 1E and F) as well as abundant starch granules. The pollen grains are spherical (diameter is about 38–42 mm) with a single operculate aperture (Fig. 1B and C). The diam- eter of the aperture and opercle is about 3.5–4.0 mm and 2.1–2.4 mm (Fig. 1C), respectively. The exine, which is about 0.7 mm wide and interspersed with granules (Fig. 1D), is composed of tectum (0.15–0.25 mm), columellae (0.2 mm) and the foot layer (0.15–0.25 mm thick). Plentiful micro- channels, with a diameter of about 0.02–0.03 mm, are present in both the tectum and foot layer (Fig. 1D). The intine is not uniformly thick (about 0.2–0.4 mm) with relatively high elec- tron density (Fig. 1D).

Figure 4.A representative 2-DEgel pattern of the prepared PRPs.

• To identify the extracellular proteins and gain an insight into protein complement of pollen, the researchers applied a two-step procedure to fractionate pollen proteins into PRPs and PIPs by eluting mature pollen grains in an isotonic buffer (see Section 2). This elution process was monitored by determining the protein concentration of each eluate, which was 2.92 mg/mL in the first eluate and decreased to lower than 0.04 mg/mL in the last eluate (Fig. 2A).The eluted pollen grains had normal I 2 -KI reaction and cell morphology (Fig. 2C) compared with fresh pollen grains (Fig. 2B), and only about 5% of them burst. They had a normal wall surface structure with some having ruptured apertures as seen under SEM (Fig. 1G and H).

Figure 5. A representative 2-DE gel pattern of the prepared pollen interior proteins (PIPs).

• Originally, each of the PRPs and PIPs was subjected to 2-DE with pH 3–10 gel strips, and the separation showed that most of the PRPs or PIPs were distributed around pH 4 to 7 (Suppl. Fig. 1A and B); therefore, the pH range strips of 4–7 were further used for the better solution of PRPs and PIPs in 2-DE. Each separation was repeated at least 3 times to ensure reproducibility of protein patterns, and the representative gel patterns of the PRPs and PIPs are shown in Figs. 4 and 5.

Labs working on this Project

• Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Research Center for Molecular & Developmental Biology, Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.
• Key Laboratory of Molecular & Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China.