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

Jinli Gong; Hang Zhang; Yunliu Zeng; Yunjiang Cheng; Xuepeng Sun; Pengwei Wang

doi:10.1186/s13007-022-00956-1

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

Plant Methods. 2022 Nov 19;18(1):124. doi: 10.1186/s13007-022-00956-1.

Authors

Jinli Gong^{1

2

3}, Hang Zhang^{2

3}, Yunliu Zeng^{2

3}, Yunjiang Cheng^{2

3}, Xuepeng Sun⁴, Pengwei Wang^{5

6

7}

Affiliations

¹ Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China.
² Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
³ National R&D Centre for Citrus Preservation, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
⁴ Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China. xs57@zafu.edu.cn.
⁵ Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. wangpengwei@mail.hzau.edu.cn.
⁶ National R&D Centre for Citrus Preservation, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. wangpengwei@mail.hzau.edu.cn.
⁷ Hubei Hongshan Laboratory, Wuhan, 430070, China. wangpengwei@mail.hzau.edu.cn.

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.

Abstract

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