Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C- terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35–HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35–HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes

Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage

The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in alpha-tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1)

Characterization of a Plastoglobule-Localized SOUL4 Heme-Binding Protein in Arabidopsis thaliana

Heme plays an active role in primary plant metabolic pathways as well as in stress signaling. In this study, we characterized the predicted heme-binding protein SOUL4. Proteomics evidence suggests that SOUL4 is a component of Arabidopsis plastoglobules (PGs, chloroplast lipid droplets). SOUL4 contains heme-binding motifs and the recombinant protein is shown here to bind heme in vitro. Fluorescence-tagged SOUL4 colocalized with the specific PG marker Fibrillin1A (FBN1A) in transiently transformed Nicotiana benthamiana leaves. In addition, SOUL4 cofractionated with another PG marker Fibrillin2 (FBN2) in sucrose gradient ultracentrifugation experiments. In vitro kinase experiments revealed that SOUL4 is phosphorylated by a yet unknown chloroplast protein kinase. Our data demonstrate that SOUL4 is a bona fide PG protein and may function in heme-buffering in the chloroplast.Peer Reviewe

Chromoplast plastoglobules recruit the carotenoid biosynthetic pathway and contribute to carotenoid accumulation during tomato fruit maturation

Tomato (Solanum lycopersicum) fruit maturation is associated with a developmental transition from chloroplasts (in mature green fruit) to chromoplasts (in red fruit). The hallmark red color of ripe tomatoes is due to carotenogenesis and accumulation of the red carotenoid lycopene inside chromoplasts. Plastoglobules (PG) are lipid droplets in plastids that are involved in diverse lipid metabolic pathways. In tomato, information on the possible role of PG in carotogenesis and the PG proteome is largely lacking. Here, we outline the role of PG in carotenogenesis giving particular attention to tomato fruit PG proteomes and metabolomes. The proteome analysis revealed the presence of PG-typical FBNs, ABC1K-like kinases, and metabolic enzymes, and those were decreased in the PG of tomato chromoplasts compared to chloroplasts. Notably, the complete β-carotene biosynthesis pathway was recruited to chromoplast PG, and the enzymes PHYTOENE SYNTHASE 1 (PSY-1), PHYTOENE DESATURASE (PDS), ZETA-CAROTENE DESATURASE (ZDS), and CAROTENOID ISOMERASE (CRTISO) were enriched up to twelvefold compared to chloroplast PG. We profiled the carotenoid and prenyl lipid changes in PG during the chloroplast to chromoplast transition and demonstrated large increases of lycopene and β-carotene in chromoplast PG. The PG proteome and metabolome are subject to extensive remodeling resulting in high accumulation of lycopene during the chloroplast-to-chromoplast transition. Overall, the results indicate that PGs contribute to carotenoid accumulation during tomato fruit maturation and suggest that they do so by functioning as a biosynthetic platform for carotenogenesis

A quantitative method to measure geranylgeranyl diphosphate (GGPP) and geranylgeranyl monophosphate (GGP) in tomato (Solanum lycopersicum) fruit

Abstract Background Isoprenoids are a very large class of metabolites playing a key role in plant physiological processes such as growth, stress resistance, fruit flavor, and color. In chloroplasts and chromoplasts, the diterpene compound geranylgeranyl diphosphate (GGPP) is the metabolic precursor required for the biosynthesis of tocopherols, plastoquinones, phylloquinone, chlorophylls, and carotenoids. Despite its key role for the plant metabolism, reports on GGPP physiological concentrations in planta have been extremely scarce. Results In this study, we developed a method to quantify GGPP and its hydrolysis product geranylgeranyl monophosphate (GGP) from tomato fruit, using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). Quantification was done by external calibration and the method was validated in terms of specificity, precision, accuracy, and detection and quantitation limits. We further demonstrate the validity of our approach by analysing GGPP contents in the ripe fruits of wild-type tomatoes and mutants defective in GGPP production. Finally, we also show that the sample preparation is key to prevent GGPP hydrolysis and mitigate its conversion to GGP. Conclusion Our study provides an efficient tool to investigate the metabolic fluxes required for GGPP supply and consumption in tomato fruit

Novel insights on the contribution of plastoglobules and reactive oxygen species to chromoplast differentiation [Dataset]

6 pages. -- The following Supporting Information is available for this article: Fig. S1. Transcript abundance of the indicated genes in GFP and crtB samples at 96 hpi. -- Fig. S2. Changes in qP and qL during chromoplastogenesis in W50 and W500 samples. -- Table S1. Accessions of N. benthamiana genes encoding isoprenoid biosynthetic enzymes. -- Table S2. Accessions of N. benthamiana genes encoding plastid proteins. -- Table S3. Primers used in this work.Peer reviewe

Proteome of tomato fruit chloroplast and chromoplast plastoglobules.

Proteome of tomato fruit chloroplast and chromoplast plastoglobules.</p

Experimental design used for tomato fruit PG proteome analysis.

PGs from mature green and red fruit were isolated separately by sucrose gradient flotation. The two independent replicate PG fractions from mature green and red fruit were analyzed by nano-liquid chromatography (nanoLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS / MS) for peptide identification. All identified peptides (Total Peptides) were further processed by Scaffold, MASCOT, and MaxQuant software to obtain corresponding individual proteins (Individual proteins). The number of individual proteins collected after merging two independent replicates (Total proteins). The Total proteins were filtered using the Uniport, TargetP, SUBA4, and TAIR databases to identify plastid protein (Plastid protein). Plastid proteins were filtered using known chloroplast and chromoplast proteome based on the existing literatures resulting in PG core proteins (PG core proteins). The new PG protein candidates (New candidates) were identified by exclusion of curated stromal, thylakoid and envelope proteins from plastid proteins (Plastid proteins) using PPDB and STRING databases as well as subcellular localization studies in the literature.</p

Tocopherols and plastoquinone were highly accumulated in the red tomato fruit.

(A) The total prenyl quinones were extracted from mature green (MG) and red (R) tomato fruit, PC-8, plastochromanol; PC-OH, hydroxy-plastochromanol; PQ-9, plastoquinone; and PQH2-9, plastoquinol were quantified (B) Quantification of phylloquinone. (C) Quantification of tocopherols. All values in the figure are the mean of 3 biological replicates (n = 3). Statistical differences were assessed with student’s t test and p values are indicated. (PDF)</p