Multi-omics Analyses Reveal The Feature Of Chromoplast Plastoglobules And Its Mechanism In Carotenoid Accumulation In Citrus Fruit

Citrus is one of the most important horticultural plants,with high economic and nutritional value.The rich and diverse carotenoids are characteristics of citrus,which play an essential role in citrus fruit quality and economic value.In the previous reports,the study of carotenoids mainly focused on the regulation mechanism of metabolic pathways,and the detection and analysis of carotenoids still used liquid chromatography(HPLC),which does not meet the requirements for in-depth study.Meanwhile,chromoplasts are a type of non-photosynthetic plastids in plants that biosynthesize and accumulate massive carotenoids in citrus.However,the characteristics of chromoplast plastoglobules(CPGs)which was an important microenvironment involved in carotenoid metabolism are unclear.Sweet orange,mandarin,and pummelo are the main materials in this study,and we systematically analyze the metabolic characteristics of carotenoids in major citrus varieties through phytochemistry,systems biology,and molecular biology.The systems biology approach(lipidome,proteome,and transcriptome)reveals the characteristics of chromoplast plastoglobules(CPG)and the mechanism involved in carotenoid metabolism,providing new insight into citrus carotenoid metabolism from the perspective of the metabolic bank.The main results of this study are as follows:1.Establishment and application of poly-terpenoid metabolomics in citrusUsing a phytochemical approach,the workflow of high-resolution mass spectrometry from extraction,separation,spectral analysis to statistical analysis of carotenoids in citrus was established.We elucidated the law of poly-terpenoid and the method of poly-terpenoid metabolomics was developed.Mass spectrometry analysis of polyterpenoids in main citrus varieties was conducted,and the bond-breaking laws of polyterpenoids in high-resolution mass spectrometry were summarized.More than 110carotenoids(including many carotenoid esters and novel carotenoids)and 23 limonoids were detected in this study.Comprehensive metabolomics analysis of carotenoids uncovered the specific characteristic carotenoids in Newhall orange,“Kaopan” pummelo,and Satsuma orange.Meanwhile,the profile of carotenoids in sweet oranges displays the characteristics of hybrid offspring.In Newhall orange,there are more carotenoids in numbers and content than those of Satsuma orange and ‘Kao Pan' pummelo.2.Separation and purification of chromoplast plastoglobules from citrus pulpIn order to obtain CPG for further research,we first established a complete workflow for isolation and purification of sweet orange CPG.The intact chromoplasts which have been preliminarily separated were quick-frozen with liquid nitrogen,and then treated at ultra-low temperature for 1.5 hours.After ultrasonic treatment,CPGs were released from chromoplasts,and the crude extract of the CPG was laid on the bottom of the centrifuge tube and then in line covered with 15%,12.5%,10%,and 5% Nycodenz gradient solution,ultracentrifugation overnight at 30,000 g and 4? to obtain the CPG floating on the top of the centrifuge tube.The fraction of the uppermost layer was found to be CPG through western blotting analysis.The membrane and a stromal fraction of chromoplasts were also isolated at the same time,distribution in the middle and bottom of the centrifuge tube.3.Comprehensive analysis of the characteristics and functions of CPGs in citrusThe purified CPGs laid the foundation for in-depth analysis and study.The various chromoplast components were obtained and analyzed by systems biology(proteome,lipidome,and transcriptome),and 44 core proteins and 35 triacylglycerols(TAGs)of CPG in citrus were defined for the first time,combined with transcriptome analysis revealed that the function of CPGs during citrus fruit development is to participate in the synthesis and metabolism.In the comparative proteomic analysis,it was found that the enzymes about carotenoid metabolism were mainly concentrated in chromoplast membrane structure,and partly in the color body matrix.The results of lipidome data analysis suggest that the main lipid substances in the plastid globules may be derived from the chromoplast membrane structure.According to the results of proteome and lipidome,we speculate that CPGs may be formed on the membrane structure inside the chromoplasts.The ELTs protein family in the core proteome of CPG could involve in the accumulation of CPG carotenoids.Among them,Cs ELT1 has significantly higher protein and transcription levels than the other four members.The subcellular localization verification results show that Cs ELT1/2/3 and PG markers were co-localization in tobacco mesophyll cells.Cs ELT1 was overexpressed in citrus callus for functional analysis,and it was found that lipids and carotenoids in the callus increased;in order to further verify whether this protein has the function of catalyzing carotenoid esters,results of experiments in yeast and engineering bacteria shows that it did not directly catalyze the formation of carotenoid esters.Cs ELT1 could promote the accumulation of carotenoids by participating in the synthesis of lipids in CPGs.In summary,we proposed a model for the formation and function of PG in the chromoplast.The formation of CPG was closely related to the chromoplast membrane.CPGs did not directly synthesize carotenoids and neutral lipids,and the main metabolites of CPG were originated from the chromoplast membrane.Thus,we suggested that the CPGs were connected to the inner membrane structure inside the chromoplast and then secreted from the membrane.Also,massive carotenoids and partial lipids that interacted with carotenoids were separated and stored in CPGs.Under certain conditions,CPGs contained massive carotenoids,and lipids were secreted from the chromoplast inner membrane,which avoids the overaccumulation of carotenoids on the membrane because carotenoids are toxic to plant cells when overaccumulation.The formation of CPG in chromoplasts could also promote efficient synthesis and storage of carotenoids.Meanwhile,CPG core proteins play a special role in performing CPG functions.In detail,the ELT protein family could increase the storage capacity of carotenoids by promoting the synthesis of lipids;the FBNs protein family may participate in the formation of CPG and the transport of carotenoids and lipids in the chromoplast.