Gong Jinli, Zhang Hang, Zeng Yunliu, Cheng Yunjiang, Sun Xuepeng, Wang Pengwei
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.
Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
Plant Methods. 2022 Nov 19;18(1):124. doi: 10.1186/s13007-022-00956-1.
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.
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.
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.
叶绿素和类胡萝卜素是植物中分布最广泛的亲脂性色素，在果实发育和成熟过程中有助于果实着色。这些色素与位于质体膜上的色素 - 蛋白质复合体组装在一起。色素 - 蛋白质复合体对多种细胞过程至关重要，然而，它们在果实中的身份和组成尚未得到表征。
通过结合使用蓝色天然聚丙烯酰胺凝胶电泳（BN - PAGE）技术和显微镜，我们研究了柑橘类果实外果皮纯化质体中的色素 - 蛋白质复合体和质体转化。采用不连续蔗糖梯度离心法从金橘果实中分离总质体，并通过显微镜观察和蛋白质免疫印迹分析评估分离质体的纯度。对不同着色阶段分离的质体进行色素自发荧光观察、蛋白质免疫印迹、二维电泳分析和BN - PAGE评估。我们的结果表明：（i）果实着色期间叶绿体分化为有色体，这种分化伴随着叶绿素/类胡萝卜素比率的降低；（ii）BN - PAGE分析揭示了柑橘类果实不同类型质体中大分子蛋白质复合体的概况；（iii）叶绿素 - 蛋白质复合体的降解速率在从叶绿体向有色体转变过程中有所不同，其稳定性一般遵循LHCII > PS II核心 > LHC I > PS I核心的顺序。
我们优化的质体分离和BN - PAGE评估方法为更好地理解植物果实中的色素 - 蛋白质复合体和质体转变提供了机会。这些尝试也有可能扩展我们对从叶绿体向有色体转变过程中蛋白质变化和色素代谢的亚细胞水平同步性的认识。
