The predicted candidates of Arabidopsis plastid inner envelope membrane proteins and their expression profiles

Abstract

Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH(2)-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of "transport facilitation," indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns.

Figure 1

Summary scheme of plastid envelope protein prediction from the Arabidopsis nuclear genome. The graph displays the number of proteins after each selection step. Subcellular localization was predicted using the TargetP version 1.01 Web server. Transmembrane region prediction was done using the TMHMM version 2.0 Web server. cTP, Chloroplast transit peptide. RC, TargetP reliability class. TM, Transmembrane domain. See “Materials and Methods” for Web site addresses for the predictors.

Figure 2

Functional classification of the Arabidopsis plastid envelope protein (AtPEM) candidates. A, Overall functional annotation status based on the short descriptions of each candidate from the Munich Information Center for Protein Sequences (MIPS; chromosomes 1, 3, and 5) and from GenBank (chromosomes 2 and 4). X, Any protein/gene/enzyme name. B, Detailed functional classification using automatically derived functional categories from the Protein Extraction Description and Analysis Tool (PEDANT) Web server (see “Materials and Methods”). C, Subclassification of the proteins classified as “transport facilitation” and “metabolism” by PEDANT.

Figure 3

Characteristics of AtPEM candidates. A, Peptide length distribution. L, Number of amino acid residues. Plastid-targeting sequences were removed based on TargetP cleavage site predictions. B, Distribution of the number of membrane-spanning domains. The TMHMM version 2.0 Web server was used for the membrane-spanning domain prediction. prob<1, Proteins with spanning domain probability less than 1.

Figure 4

Digital differential display analysis. AtPEM candidates with statistically different levels (P < 0.005, where P is probability of difference being by chance) of EST frequencies are displayed. Number of ESTs in the “flowers,” “roots,” or “seeds” pools was compared with that in the reference “mixed” library pool. The bars indicate the abundances of the ESTs (EST frequency axis) corresponding to the proteins (proteins axis) in each library pool (tissue axis).

Figure 5

Tissue-specific microarray analysis of AtPEM candidates. AtPEM candidates with >2-fold tissue-specific expression in at least two different tissue comparisons are shown clustered according to the tissue specificity (indicated with the brackets and labeled). The intensity of green and red colors represents the relative level of transcripts (refer to the color keys at the bottom for the actual ratios) in tissues under comparison (the tissues under comparison are indicated above the image and colored with representative colors). The data for flower, leaf, and root comparisons were from SMD (http://genome-www.stanford.edu/microarray) and data for seed versus leaf and seed versus plantlet were from the Arabidopsis developing seed array (Girke et al., 2000; Ruuska et al., 2002). The graphic was generated by Tree View software (Eisen et al., 1998).