Chapter 4
Isolation of Apoplastic Fluid from Woody Plant Leaves:
Grapevine and Coffee as a Case Study
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es
Abstract
Proteomics is one of the key approaches to understand plant cell physiology involving the regulation of
expression of many genes and metabolite production. Technical advances allowed a deeper characterization
of plant proteomes, highlighting the need to study cellular compartments. The apoplast is the cellular
compartment external to the plasma membrane including the cell wall, where a broad range of processes
take place including intercellular signaling, metabolite transport, and plant–microbe interactions. Due to
the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves while maintaining cell
integrity, which is particularly true for woody plants. Here, we describe the vacuum infiltrationcentrifugation (VIC) method for the extraction of the apoplastic fluid compatible with high-throughput
proteomic approaches and biochemical analysis from different woody plants.
Key words VIC method, Apoplast, Apoplastic fluid, Grapevine leaves, Coffee leaves, Vitis vinifera,
Coffea arabica, Electrophoresis, Concentration
1 Introduction
The plant extracellular space, or apoplast, is a highly dynamic
compartment affected by environmental conditions and participates in plant signaling, defense, growth, physiology, cell wall
maintenance, and reproduction [1]. It is defined by the plant cell
wall, extracellular matrix, and the intercellular spaces where the
apoplastic fluid (APF) circulates [2]. APF studies allow uncovering
plant’s secretome. It comprehends a set of proteins and small
molecules exported out of the symplast that play several roles,
where the response to biotic and abiotic stresses takes a spotlight
position [3–5].
Apoplastic fluid extraction from plant systems is far from easy
and remains challenging. This is particularly true for recalcitrant
plants, as the majority of them are woody plants. Thus, the study of
apoplast dynamics is hindered by low protein abundance and high
possibility of damaging the plant cell, leading to cytoplasmic
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
49
Isolation of Apoplastic Fluid from Woody Plant Leaves:
Grapevine and Coffee as a Case Study
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es
Abstract
Proteomics is one of the key approaches to understand plant cell physiology involving the regulation of
expression of many genes and metabolite production. Technical advances allowed a deeper characterization
of plant proteomes, highlighting the need to study cellular compartments. The apoplast is the cellular
compartment external to the plasma membrane including the cell wall, where a broad range of processes
take place including intercellular signaling, metabolite transport, and plant–microbe interactions. Due to
the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves while maintaining cell
integrity, which is particularly true for woody plants. Here, we describe the vacuum infiltrationcentrifugation (VIC) method for the extraction of the apoplastic fluid compatible with high-throughput
proteomic approaches and biochemical analysis from different woody plants.
Key words VIC method, Apoplast, Apoplastic fluid, Grapevine leaves, Coffee leaves, Vitis vinifera,
Coffea arabica, Electrophoresis, Concentration
1 Introduction
The plant extracellular space, or apoplast, is a highly dynamic
compartment affected by environmental conditions and participates in plant signaling, defense, growth, physiology, cell wall
maintenance, and reproduction [1]. It is defined by the plant cell
wall, extracellular matrix, and the intercellular spaces where the
apoplastic fluid (APF) circulates [2]. APF studies allow uncovering
plant’s secretome. It comprehends a set of proteins and small
molecules exported out of the symplast that play several roles,
where the response to biotic and abiotic stresses takes a spotlight
position [3–5].
Apoplastic fluid extraction from plant systems is far from easy
and remains challenging. This is particularly true for recalcitrant
plants, as the majority of them are woody plants. Thus, the study of
apoplast dynamics is hindered by low protein abundance and high
possibility of damaging the plant cell, leading to cytoplasmic
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
49