IC4R009-Proteomic-2007-17385905

Project Title

• Proteome and Phosphoproteome Differential Expression under Salinity Stress in Rice (Oryza sativa) Roots

The Background of This Project

• Salinity stress is a major abiotic stress that limits agriculture productivity worldwide. Rice is a model plant of monocotyledons, including cereal crops. Studies have suggested a critical role of protein phosphorylation in salt stress response in plants. However, the phosphoproteome in rice, particularly under salinity stress, has not been well studied.

Plant Culture & Treatment

• Rice seeds (Oryza sativa, cv. Nipponbare) were germinated at 25 °C in Hoagland solution.27 After 20 days, seedlings were treated with 150 mM NaCl in Hoagland solution and the roots were harvested at 0,10, and 24 h of treatment.

Protein Extraction and 2-D PAGE

• Rice root tissues were ground in liquid nitrogen with mortar and pestle into fine powders.Proteins were extracted using a modified phenol extraction protocol28 as follows: Ground tissues were suspended in an extraction buffer (0.9 M sucrose, 0.5 M Tris-HCl, 0.05 M EDTA,0.1 M KCl, and 2% â-mercaptoethanol added freshly, the final pH was 8.7), were mixed with an equal volume of saturated phenol (pH 8.0), and then were homogenized for 10 min. The homogenate was centrifuged at 2500g for 10 min, the phenol phase was recovered, and the phenol extraction was repeated three times. The final collection of phenol was mixed with five volumes of precipitation buffer (methanol with 0.1 M ammonium acetate and 1% â-mercaptoethanol). Precipitation was carried out at -70 °C overnight. The precipitant was recovered by centrifugation at 13 400g for 10 min, and the pellet was washed three times with cold precipitation buffer and another three times with ice cold 70% ethanol. The protein pellet was lyophilized to powder in a speed vacuum (LABCONCO, model LYPH-LOCK 6) and was stored at -70 °C. At least three biological replicas were extracted for each treatment.
• Proteins were dissolved in rehydration buffer (7M urea, 2 M thiourea, 4% CHAPSO, 1% dithiotreitol (DTT), and0.2% ampholines) and were centrifuged at 12 000g for 10 min to remove undissolved content. The supernatant was quantified using a Bio-Rad Rc Dc protein assay kit according to the instructions of the manufacturer. The quantified proteins were then used for 2D PAGE. Isoelectric focusing (IEF) was carried out using a Bio-Rad PROTEAN IEF cell on 24-cm 3-10 pH nonlinear IPG strips (Bio-Rad). One milligram of protein in 400µL of rehydration buffer was loaded into the IEF tray and active rehydration was carried out at 23 °C for 12 h, followed by 250 V for 2 h, and a linear increase of voltage to 10 000 V for 4 h,and the isoelectric focusing was performed at 23 °C for a total of 90 000 VH. After the completion of IEF, the strips were equilibrated in an equilibrating buffer containing 6 M urea,0.375 M Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, and 2% dithiothreitol for 15 min followed by equilibration for another 15 min in a buffer containing 6 M urea, 0.375 M Tris-HCl (pH6.8), 20% glycerol, 2% SDS, 0.1% bromophenol blue, and 2.5%iodoacetamide.The equilibrated IPG strips were loaded on horizontal slab gels (25 × 20.5 × 1.5 mm) containing 12% (w/v) separating gel and 4% stacking gel (w/v). Electrophoresis was carried out in a Bio-Rad PROTEAN PLUS horizontal Dodeca cell at 20 mA/gel.The gels were stained with SYPRO Ruby fluorescence stain(Bio-Rad) according to the protocols provided by the manufacturer and were scanned with a VersaDoc4000 image system(Bio-Rad). The images were analyzed with PDQUEST 7.4.0. software (Bio-Rad, Hercules, CA). For each treatment, at least three 2-DE gels representing the three biological replicas were used for data analyses. The cutoff for differentially regulated proteins was 2-fold of change in all biological replicas.

Research Findings

• Pro-Q Diamond Phosphoprotein in Gel Stain is a fluorescence dye specific for phosphoproteins. To examine the phosphoproteome and proteome in rice roots, the researchers stained proteins from 20-day-old rice roots with Pro-Q Diamond dye and SYPRO Ruby dye, respectively, following protein separation on 2-DE gels. As shown in Figure 1A, the majority of the Pro-Q Diamond dye-stained protein spots had a relatively low pI, and it was very obvious for those heavily stained protein spots. In contrast,the SYPRO Ruby stained proteins were more evenly distributed on the 2-DE gel (Figure 1B). Many protein spots that were heavily stained with SYPRO Ruby were not stained or were weakly stained by Pro-Q Diamond dye. Conversely, many spots that were weakly stained by SYPRO Ruby were heavily stained by Pro-Q Diamond dye as revealed by comparing the spots labeled in Figure 1C and 1D.

'Figure 1. 2-DE gel images of rice root proteome revealed by Pro-Q Diamond in Gel Stain and SYPRO Ruby fluorescence stain. Proteins were extracted from 20-day-old rice roots, were separated on 2-DE gels, and were stained with Pro-Q Diamond dye and SYPRO Ruby.(A) 2-DE gel image revealed by Pro-Q Diamond in Gel stain. (B) 2-DE gel image revealed by SYPRO Ruby stain. (C) An enlarged section of the image shown in A. The spot positions of several major proteins are marked and labeled for comparison. (D) An enlarged section of the image shown in B. The spot positions of several major proteins are marked and labeled for comparison.'

• To study how the putative phosphoproteins stained by Pro-Q Diamond dye resrpond to NaCl stress, we examined the rice root proteome with the Pro-Q Diamond dye after 10 and 24 h of 150 mM NaCl treatments.Twenty Pro-Q Diamond dye-stained protein spots displayed over 2-fold upregulation and 18 protein spots displayed over 2-fold downregulation in all three biological replicas (Figure 2 and data not shown).

'Figure 2. Protein differential regulation under salinity stress revealed by Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were extracted from 20-day-old control and salt-treated rice roots,were separated on 2-DE gels, and were stained with Pro-Q Diamond dye. The cutoff of differential regulation was 2-fold of change in three biological replicas. (A) Putative phosphoproteome image of rice roots without salt stress. All protein spots subjected to upregulation under salinity stress are marked with black arrows and all protein spots subjected to downregulation are marked with white arrows. (B) Putative phosphoproteome image after 10 h of NaCl (150 mM) treatment. Proteins upregulated after 10 h of salt treatment are marked with black arrows and the proteins downregulated after 10 h of salt treatment are marked with white arrows. (C) Putative phosphoproteome image after 24 h of NaCl (150 mM) treatment. Proteins upregulated after 24 h of salt treatment are marked with black arrows and the proteins downregulated after 24 h of salt treatment are marked with white arrows.

• Protein differential expression in rice roots, after 10 and 24 h of salt treatment, was also examined using SYPRO Ruby stain following 2-DE separation.Thirty-one protein spots displayed over 2-fold upregulation and 13 protein spots displayed over 2-fold downregulation in all three biological replicas (Figure 3 and data not shown).

'Figure 3. Protein differential expression under salinity stress revealed by SYPRO Ruby stain. Proteins were extracted from 20-day-old rice roots, were separated on 2-DE gels, and were stained with Pro-Q Diamond dye. The cutoff of differential regulation was 2-fold of change in three biological replicas. (A) Rice root proteome image without salt stress. All protein spots subjected to upregulation under salinity stress are marked with black arrows and all protein spots subjected to downregulation are marked with white arrows. (B) Rice root proteome image after 10 h of NaCl (150 mM) treatment. Proteins upregulated after 10h of salt treatment are marked with black arrows and the proteins downregulated after 10 h of salt treatment are marked with white arrows. (C) Rice root proteome image after 24 h of NaCl (150mM) treatment. Proteins upregulated after 24 h of salt treatment are marked with black arrows and the proteins downregulated after 24 h of salt treatment are marked with white arrows.'

• After MALDI-TOF/TOF analyses, 23 of the 31 upregulated proteins and 8 of the 11 downregulated proteins had been identified

with high confidence as shown in Table 2.

'Table 2. Differentially Expressed Proteins in the Time Course of Salt Stressa'

• As shown in Figure 4, seven proteins displayed differential expression whether the gel was stained by Pro-Q Diamond dye or SYPRO Ruby. The other differentially regulated proteins were specific either to Pro-Q Diamond stain or SYPRO Ruby stain.These results suggest that most proteins with quantity change during salinity stress are not phosphoproteins or have no change in phosphorylation level. On the other hand, most differentially regulated phosphoproteins have modification level change instead of quantity change during salinity stress.

'Figure 4. Venn diagram analyses of the differentially upregulated proteins under salinity stress. Differentially upregulated proteins revealed by SYPRO Ruby and Pro-Q Diamond Phosphoprotein Gel Stain were analyzed.'

Labs working on this Project

• Department of Biochemistry and Molecular Biology, Mississippi State University,Mail box 9650, Mississippi State, Mississippi 39762

Corresponding Author

• Zhaohua Peng:zp7@ra.msstate.edu.