Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in c4-related metabolite fluxes and development

Abstract

As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second "green revolution" will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2-15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast development.

Keywords: C4 plant; bundle sheath cells; chloroplast envelope proteins; mesophyll cells; photosynthesis.

Figure 1

Distribution of envelope proteins throughout different cell compartments (A) and by membrane association (B).

Figure 2

Differences in the relative abundance of proteins based on the bundle sheath: mesophyll ratio of their spectral ion counts. Data are based on values shown in Tables S3 and S4 in Supplementary Material. Except for known controls, only membrane-associated proteins are integrated. Due to space constrains only ca. 33% of the bars are labeled. The complete protein list and figure are shown in Table S4 in Supplementary Material. Values are the averages from two mesophyll and three BS data sets. Red bars indicate proteins used for further studies.

Figure 3

RT-PCR showing relative abundance of transcripts for several genes encoding chloroplast envelope proteins. Band intensities of three to five biological replicates were quantified and are displayed in Table 2. MS, mesophyll; BS, bundle sheath.

Figure 4

Localization of chloroplast envelope proteins with C-terminal GFP or YFP tags that were transiently expressed in Nicotiana tabacum. The first column shows chlorophyll autofluorescence, the second column GFP or YFP fluorescence, column three shows the overlay. Protein names (as used in Table 1) are indicated on the right of each row.

Figure 5

Quantification of semiquantitative RT-PCR showing tissue-specific expression of genes encoding the 5TM protein (green bars), ER-AP (yellow bars), PIC-like protein (blue bars), and UP-d (red bars). Representative gel pictures are shown in the lower half. Band intensities of three to five biological replicates were quantified and displayed in the bar graph.

Figure 6

Change in the expression of genes encoding the PIC-like protein (A), ER-AP (B), 5TM protein (C), and UP-d (D). Values were obtained from five to six biological replicates.