Chromoplast-specific carotenoid-associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits

Abstract

Tomato (Solanum lycopersicum) high-pigment mutants with lesions in diverse loci such as DNA Damage-Binding Protein1 (high pigment1 [hp1]), Deetiolated1 (hp2), Zeaxanthin Epoxidase (hp3), and Intense pigment (Ip; gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and wild-type (cv Ailsa Craig) fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast-specific carotenoid-associated protein (CHRC) in hp1 fruits at red-ripe stage that is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper (Capsicum annuum) revealed a 2-fold increase in the abundance of CHRC protein in the red-ripe stage of hp1 fruits compared with the wild type. CHRC levels in hp2 were found to be similar to that of hp1, whereas hp3 and Ip showed intermediate levels to those in hp1, hp2, and wild-type fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, Zeaxanthin Epoxidase, and Ip up-regulates CHRC levels. Increase in CHRC levels may contribute to the enhanced carotenoid content in these high-pigment fruits by assisting in the sequestration and stabilization of carotenoids.

Figure 1.

Characterization of the phenotypic and metabolic differences between wild-type and hp1 fruits. A, Phenotypes of wild-type (WT) and hp1 fruits at MG, BR, and RR stages during ripening. B, Levels of lycopene (left) and β-carotene (right) in RR fruits of the wild type (WT) and hp1 (n > 3). C, Lutein levels in RR fruits of hp1 and the wild type (WT; n > 3). D, Ethylene emission from wild-type (WT) and hp1 fruits at different stages of ripening (n > 3). FW, Fresh weight. Error bars indicate se. E, Comparison of pericarp proteome profiles isolated from wild-type and hp1 fruits at MG, BR, and RR stages. Representative images from two-dimensional gel electrophoresis are shown. [See online article for color version of this figure.]

Figure 2.

A representative two-dimensional gel electrophoresis colloidal Coomassie blue-stained gel showing the profile of proteins extracted from hp1 fruits at the BR stage. The numbers indicate differentially expressed proteins that were later identified by mass spectrometry, and the arrows indicate their relative positions on the gel.

Figure 3.

Hierarchical clustering analysis of differentially expressed proteins in wild-type (WT) and hp1 fruits during ripening. A, Using PermutMatrix 1.9.3, proteins were clustered into three clusters based on their percentage spot volumes. A color bar corresponding to the relative levels of expression is given at the top of the heat map. The identity of each protein (spot no.) is indicated to the right of the rows. The columns represent levels of proteins at MG, BR, and RR stages of wild-type and hp1 fruits. B, Pie diagram showing the relative distribution of different functional classes of proteins in each cluster. [See online article for color version of this figure.]

Figure 4.

The biosynthetic pathway of carotenoids depicting the expression profiles of enzymes catalyzing different steps beginning from the precursors. Dotted arrows indicate multiple steps in the pathway. The transcripts were quantified in the wild type and hp1 at MG, BR, and RR stages of fruit development using real-time PCR. The transcripts were expressed after normalization with two internal controls, β-actin and ubiquitin. GA3-P, Glyceraldehyde-3-phosphate; DXP, deoxy-xylulose 5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GGDP, geranylgeranyl diphosphate; DXS, deoxy-xylulose 5-phosphate synthase; IDI, inositol diphosphate synthase; GGDS2, geranylgeranyl diphosphate synthase2; PSY1, phytoene synthase1; PSY2, phytoene synthase2; PDS, phytoene desaturase; ZIS, ζ-carotene isomerase; CRTISO, carotenoid isomerase; ZDS, ζ-carotene desaturase; LCYB1, lycopene β-cyclase1; LCYB2, lycopene β-cyclase2; CYCB, chromoplast-specific lycopene β-cyclase; LCYE, lycopene ε-cyclase; CRTRB2, β-carotene hydroxylase2; ZEP, zeaxanthin epoxidase; VDE, violoxanthin deepoxidase; NCED1, 9-cis-epoxycarotenoid dioxygenase1; CRTRB1, β-carotene hydroxylase1; CYP97A29, cytochrome P450 carotenoid β-hydroxylase A29; CYP97C11, cytochrome P450 carotenoid ε-hydroxylase C11; WT, wild type.

Figure 5.

Comparison of CHRC levels in wild-type (WT) and hp1 fruits. Unless specifically indicated, equal amounts of protein were loaded on all the immunoblots. A, Relative spot volume of CHRC protein in wild-type and hp1 fruits at different stages of ripening. B, Real-time PCR quantification of CHRC transcript in wild-type and hp1 fruits at different stages of ripening. C, Immunoblotting of proteins from bell pepper and tomato fruits. The polyclonal antibody raised against bell pepper PAP recognized a protein of 35 ± 2 kD in tomato. Since PAP levels were very high in bell pepper, a reduced amount of protein (0.3 μg) was loaded compared with wild-type tomato fruits (10 μg). D, Comparison of CHRC levels in wild-type and hp1 fruits at the RR stage. Progressively increasing concentrations of proteins were loaded on the gel. The equivalence in the band intensity of hp1 (2.5 μg) with that of the wild type (5.0 μg) indicates that the CHRC level in hp1 is approximately 2-fold higher than in the wild type. E, Comparison of CHRC levels in wild-type and hp1 fruits at MG and RR stages. Ripening stimulated an increase in the level of CHRC protein from MG to RR stages, with higher CHRC levels in hp1 fruits. F, Comparison of CHRC levels in RR fruits of AC, cv Arka vikas (AV), and nor. Note that the nor mutant, although it accumulates very little lycopene, exhibits a normal level of CHRC equal to the other two cultivars. G, Relative abundance of CHRC protein in hp1 and hp2 fruits at the MG stage. H, Relative levels of CHRC in hp1, hp2, and Ip fruits at the RR stage. Note that hp1 and hp2 exhibit similar levels of CHRC, while Ip shows an intermediate level of CHRC between the wild type and hp1 and hp2 fruits. I, Analysis of CHRC levels in RR fruits of the wild type, hp3, and hp1. The order of abundance of CHRC is hp1 > hp3 > wild type. J, Comparison of CHRC transcript levels in RR fruits of hp1, hp2, hp3, and Ip in relation to the wild type. Note that all the mutants exhibit high CHRC transcript level compared with the wild type.

Figure 6.

Confocal microscopy imaging and western blotting of isolated plastoglobules. A, Suc density gradient purification of plastoglobules (PG). Note that the distinct upper layer of purified plastoglobules is well separated from the underlying chromoplast membranous fractions. OM/IM indicate outer and inner membranes of chromoplasts. B to D, Purified plastoglobules were visualized using a confocal microscope. On excitation with a 488-nm laser, the plastoglobules exhibit strong green fluorescence (emission between 500 and 510 nm; B) but show negligible red fluorescence (emission between 740 and 750 nm; C). D shows a bright-field image of the plastoglobules. E, Enlarged view of a single plastoglobule. Note that the green fluorescence emitted from plastoglobules is uniformly distributed. F, Absorption spectrum of the extract obtained from purified plastoglobules showing characteristic carotenoid peaks at 440 to 475 nm. G, Western analysis of CHRC levels in the purified plastoglobule fraction (PG; 0.33 μg) in comparison with the chromoplast membrane fraction (CF [the chromoplast fraction prior to sonication]; 2.5 μg) and total protein from the outer pericarp of RR fruits of hp1 (FE, fruit extract; 2.5 μg). Note that the plastoglobule fraction shows enrichment of CHRC protein. [See online article for color version of this figure.]