Figueiredo Joana, Cavaco Ana Rita, Guerra-Guimarães Leonor, Leclercq Céline, Renaut Jenny, Cunha Jorge, Eiras-Dias José, Cordeiro Carlos, Matos Ana Rita, Sousa Silva Marta, Figueiredo Andreia
University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisbon, Portugal.
Laboratório de FTICR e Espectrometria de Massa Estrutural, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal.
Physiol Plant. 2021 Mar;171(3):343-357. doi: 10.1111/ppl.13198. Epub 2020 Sep 17.
The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.
对复杂生物系统的分析一直是研究人员面临的挑战。系统生物学的主要目标是通过整合来自大规模分析方法（包括转录组学、蛋白质组学和代谢组学）以及更专业的“组学”（如表观基因组学和脂质组学）的数据集，来破译细胞内的相互作用。研究不同的细胞区室有助于更全面地了解细胞动态。植物质外体是细胞膜外部的细胞区室，包括细胞壁，其分析尤为困难。尽管我们了解质外体参与了从细胞生长到应激反应的多个过程，但由于缺乏适用于每种植物系统的有效提取方法，其动态仍知之甚少。分析葡萄等木本植物带来了更多挑战。葡萄是全球最重要的水果作物之一，对其质外体进行更广泛的表征对于深入了解其生理学和细胞机制至关重要。在这里，我们首次描述了一种真空渗透离心法，该方法可以从单个样品的叶片中同时提取葡萄质外体蛋白和代谢物，适用于高通量质谱分析。从两个葡萄品种“特林卡迪拉”和“丽晶”中提取的质外体直接用于蛋白质组学和代谢组学分析。通过纳升液相色谱-串联质谱对蛋白质组进行分析，鉴定出700多种常见蛋白质，其生物学功能高度多样。通过傅里叶变换离子回旋共振质谱获得的代谢组图谱鉴定出514种独特的假定化合物，揭示了广泛的分子类别。
