Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes

Abstract

Plastoglobules (PGs) are oval or tubular lipid-rich structures present in all plastid types, but their specific functions are unclear. PGs contain quinones, alpha-tocopherol, and lipids and, in chromoplasts, carotenoids as well. It is not known whether PGs contain any enzymes or regulatory proteins. Here, we determined the proteome of PGs from chloroplasts of stressed and unstressed leaves of Arabidopsis (Arabidopsis thaliana) as well as from pepper (Capsicum annuum) fruit chromoplasts using mass spectrometry. Together, this showed that the proteome of chloroplast PGs consists of seven fibrillins, providing a protein coat and preventing coalescence of the PGs, and an additional 25 proteins likely involved in metabolism of isoprenoid-derived molecules (quinines and tocochromanols), lipids, and carotenoid cleavage. Four unknown ABC1 kinases were identified, possibly involved in regulation of quinone monooxygenases. Most proteins have not been observed earlier but have predicted N-terminal chloroplast transit peptides and lack transmembrane domains, consistent with localization in the PG lipid monolayer particles. Quantitative differences in PG composition in response to high light stress and degreening were determined by differential stable-isotope labeling using formaldehyde. More than 20 proteins were identified in the PG proteome of pepper chromoplasts, including four enzymes of carotenoid biosynthesis and several homologs of proteins observed in the chloroplast PGs. Our data strongly suggest that PGs in chloroplasts form a functional metabolic link between the inner envelope and thylakoid membranes and play a role in breakdown of carotenoids and oxidative stress defense, whereas PGs in chromoplasts are also an active site for carotenoid conversions.

Figure 1.

Purification scheme and photograph of purified PGs of Arabidopsis chloroplasts (in color).

Figure 2.

1-D PAGE analysis of PG proteomes from Arabidopsis chloroplasts (A), pepper chromoplasts (B), and low-density lipid structures from rice etioplasts (B). Labeling of the gel lanes is as follows: R2, ClpR2; C, wild-type plant grown under optimal conditions; HL, wild-type plant grown for 7 d at HL (1,500 μE m⁻² s⁻¹); D, wild-type plants kept for 7 d in total darkness; P, PGs isolated from red pepper chromoplasts; R, low-density lipid structures from rice etioplasts. Proteins were identified in the numbered bands; accession numbers are listed in Tables I and II (Arabidopsis), and Table III (pepper). Detailed results for MALDI-TOF MS peptide mass fingerprinting on chloroplast PGs are provided in Supplemental Table I.

Figure 3.

Outline of differential accumulation analysis using stable-isotope labeling with DCDO and HCHO. For a pairwise comparison of dark and HL samples, 2.5 μg of each digested proteome were labeled with DCDO or HCHO. Each quantification was repeated with a switch of isotope labels so as to minimize possible isotope biases on the quantification (A). Comparison between the different stress treatments (dark and HL) and the unstressed condition were always pairwise (B). C, Detailed m/z isotope envelopes for peptide GDGGLFVLAR labeled with HCHO (mass shift of 28 D per free amine) or DCDO (mass shift of 32 D per free amine).

Figure 4.

Schematic overview of proposed organization (A) and functional role of the PG and its proteome (B). PGs consist of a monolayer of lipids and sequester different hydrophilic small molecules, such as quinones and tocopherols. Structural proteins (fibrillins) and enzymes are attached to or embedded in the monolayer, but proteins lack transmembrane domains (A). Integration of PG functions in plastid metabolism (B).