Abstract
Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C(4) photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.
Highlights
Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C4 photosynthesis
We published a quantitative analysis of purified M and BS chloroplast stromal proteomes in which BS-M protein accumulation ratios for 125 accessions were determined; this covered a limited range of plastid functions, it enabled the integration of information from previous studies (Majeran et al, 2005)
We identified 1,662 protein models corresponding to 954 protein accessions when counting only one model per gene
Summary
Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C4 photosynthesis. The conclusions of these proteome analyses are summarized by Majeran and van Wijk, 2009 Whereas these proteomics studies provide significant progress in understanding the organization of C4 metabolism in maize, three aspects have not been adequately addressed: (1) the stromal proteomes of BS and M chloroplasts likely each contain more than 1,500 proteins, but the BS-M ratios for only approximately 125 proteins were quantified, resulting in very limited coverage of several important secondary metabolic pathways such as sulfur, fatty acid, amino acid, and nucleotide metabolism; (2) information about relative concentrations of stromal proteins in BS and M chloroplasts is lacking but is needed as a basis for quantitative modeling and metabolic engineering of C4 photosynthesis and other metabolic pathways; the growing “toolbox” of proteomics and MS allows for such quantitative analyses (Bantscheff et al, 2007; Kumar and Mann, 2009); (3) the soluble (Majeran et al, 2005) and membrane (Majeran et al, 2008) proteome data sets were analyzed by different techniques and mass spectrometers, mostly due to the improvement of commercial mass spectrometers in that time frame.
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