Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

Abstract

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.

Keywords: MudPIT; callus; cell wall proteins; proteomics; rice.

Fig. 1.

Experimental scheme for extraction of tightly-bound rice cell wall proteins. (A) Rice callus culture was maintained on NB medium. (B) Rice calli after homogenization in a blender, followed by grinding using a mortar and pestle; note the presence of remaining intact tissue. (C) Complete cellular disruption achieved following French press treatment. (D) Extracted cell wall proteins separated on 1-D SDS-PAGE: M, molecular marker; Lp, leaf pellet; Ls, leaf soluble fraction; Cp, callus pellet; Cs, callus soluble fraction (cytosolic proteins in supernatant); Ca, CaCl₂-extracted cell wall proteins derived from Cp; SDS, SDS-extracted proteins from pellet after CaCl₂ extraction. (E, F) Western blot analyses performed to evaluate the purity of extracted cell wall proteins. Actin (E): cytosolic and PD marker, BiP (F): ER marker. (G) An MS/MS spectrum conducted to identify rice cell wall proteins.

Fig. 2.

Venn diagrams illustrating the populations of rice proteins identified using CaCl₂ and SDS extraction methods. (A) CaCl₂-extracted proteins were separated and processed based on the scheme presented in Fig. 1. CaCl₂-H and CaCl₂-L; total number of proteins identified within the high (H) and low (L) molecular weight fractions. (B) SDS-extracted proteins were separated and processed based on the scheme presented in Fig. 1. SDS-H and SDSL; total number of proteins identified within the high (H) and low (L) molecular weight fractions. (C) Comparison between rice proteomes extracted by CaCl₂ and SDS methods. A total of 1999 non-redundant rice callus proteins were identified using this sequential two-step extraction protocol.

Fig. 3.

Statistics of rice cell wall proteins containing a predicted signal peptide and transmembrane domains. (A) Number of proteins having a predicted signal peptide based on SignalP 3.0 using the NN and HMM algorithms. (B) Number of proteins having predicted transmembrane domains (TMDs) based on the TMHMM v. 2.0 program.

Fig. 4.

Pie diagram illustrating the potential sub-cellular protein localization for the rice callus proteome. Classical cell wall proteins contain a signal peptide (predicted by both NN and HMM algorithms of SignalP 3.0), but lack transmembrane domains (predicted by the TMHMM v. 2.0 program). Plasma membrane proteins were predicted by the TMHMM v. 2.0 program. Proteins possessing both a predicted signal peptide and a transmembrane domain were scored as classical cell wall proteins. Plastid and mitochondrial proteins were predicted by the TargetP 1.1 program and considered as contaminated proteins. Non-classical cell wall proteins were predicted by the SecretomeP 2.0 program. Proteins containing an ER retention motif (KDEL or HEDL) at C-terminal region were identified as ER proteins.

Fig. 5.

Venn diagram illustrating GPI-anchored proteins predicted by the Big-PI, Pred-GPI, and GPI-SOM programs

Fig. 6.

Conserved domains and functional categories for the 389 classical rice cell wall proteins. (A) Distribution of the most frequently identified conserved domains based on the InterProScan database implemented in Blast2GO. Conserved domains with more than two proteins are displayed. (C) Functional categories identified based on the WallProtDB database (http://www.polebio.scsv.upstlse.fr/WallProtDB/).

Fig. 7.

Distribution of rice cell wall proteins identified by four methods. (A) Weakly-bound cell wall proteome (Chen et al., 2009). (B) Rice proteins identified by secretome analysis (Cho et al., 2009). (C) Tightly-bound cell wall proteins extracted by CaCl₂ treatment. (D) Tightly-bound cell wall proteins extracted by SDS treatment.

Fig. 8.

Comparison of rice and Arabidopsis classical cell wall proteins. BLASTP was used to analyze 496 rice and 244 Arabidopsis cell wall proteins (CWPs; cutoff value of 1E-6).