Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

Abstract

Background: Chlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized.

Results: By using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core.

Conclusions: Our optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts.

Keywords: BN-PAGE; Carotenoid; Chlorophyll; Citrus fruit; Pigment-protein complex; Plastid.

Fig. 1

Workflow of plastid isolation and purification from the peel of kumquat fruit using sucrose density gradient centrifugation

Fig. 2

Plastids isolated from the outer peel of kumquat at three different coloring stages. A Representative photographs of kumquat fruit used for plastid isolation. From left to right: ‘Green’, ‘Breaker’ and ‘Orange’ represent the stage from immature green to mature orange phase. B Frozen sections of epicarp. C Transmission electron microscopic (TEM) images showing the ultrastructural features of plastids in the outer peel of kumquat. Red arrow shows thylakoid lamellar structure. Yellow arrow shows plastoglobule (PG). Blue arrow shows starch grains. D Statistical analysis of the size of PGs per plastid in flavedo at three different coloring stages from Fig. 2, C. The y-axis represents the number of PGs at a given size, which indicated on the x-axis. The statistics was performed for a total of 140 PGs from 15 plastids. (E) Statistical analysis of the average number of PGs per plastid in flavedo at different maturation stages. The statistics was calculated from 15 plastids selected randomly

Fig. 3

Plastid isolation and purity assessment. A Separation of plastids on a discontinuous sucrose gradient (0.75, 0.92, 1.20, 1.50 M) at three different coloring stages. B Light microscopy of purified chloroplasts from band 2 (I), plastid fractions were designated as bands 1, 2, 3, 4 from the top to the bottom of the sucrose gradient (II). C Assessment on the purity of isolated plastids using immunoblots. Purified plastids (band 2) were detected using antibodies for Tic110 (translocon at the inner envelope membrane of chloroplasts) and cytosolic UGPase. Each lane was loaded with 20 µg of total protein. The positions of the molecular markers are indicated on the left. Chlo, chloroplast; Chlo-Chro, intermediate plastids between chloroplasts and chromoplasts; Chro, chromoplast. D Coomassie blue protein profiles of purified plastids (band 2) from kumquat peel as compared with total kumquat peel proteins

Fig. 4

Confocal images of purified plastids and analysis of pigment content. Confocal images of chloroplasts A, intermediate plastids (B) and mature chromoplasts (C) suspensions isolated from green, breaker and fully mature pericarp of kumquat, respectively. Images are overlays of chlorophyll autofluorescence emitted at 650–700 nm (red) and carotenoid autofluorescence emitted at 500–600 nm (green), respectively. The extracts mainly containing chlorophyll appear red, those only containing carotenoids appear green, and those containing both chlorophyll and carotenoids appear yellow. Bar, 25 μm. The excitation wavelength was 488 nm. The peaks of fluorescence emission around 520–550 nm and 680 nm correspond to carotenoids and chlorophylls in citrus fruit, respectively. Measurement of chlorophyll concentration (D), chlorophyll/carotenoid ratio (E) and carotenoid concentration (F) in kumquat fruit at green, breaker and orange color stages, respectively. The pigment concentration is recorded with the unit of μg g ⁻¹ fresh weight

Fig. 5

Blue-native PAGE profile and immunoblot with antibodies against photosynthesis-related proteins from isolated plastids at green, breaker and orange color stages. A Blue-native PAGE profile of chloroplast membrane protein complexes at green stage. Molecular mass markers are indicated on the left of the figure. B Separation of membrane protein complexes from chloroplasts (left lane), chlo-chromoplasts (middle lane) and chromoplasts (right lane) by BN-gel electrophoresis. Red asterisks indicate the bands present in all three periods. Each lane of BN-gel was loaded with 200 μg of protein. Immunoblotting analysis showing changes in the relative amounts of protein subunits in PS I complexes (C) and PS II complexes (D). Each lane of immunoblotting analysis was loaded with 20 μg of protein

Fig. 6

Model of the chloroplast-to-chromoplast differentiation process during fruit ripening, accompanied by disruption of the chlorophyll-photosynthetic (PS) protein complexes and extensive synthesis of the carotenoid-non-PS protein complexes