methods have been described to successfully establish them [5, 6],
and the advantages of working with Arabidopsis cell cultures containing functional chloroplasts have become increasingly evident
[5, 7].
Cell suspension cultures can be used for a number of structural
and functional studies, including signaling ones. The use of Arabidopsis photosynthetic and heterotrophic cell suspension cultures
has recently highlighted differences between non-green plastids
and chloroplasts in terms of organellar calcium signals in response
to environmental stimuli [8]. Moreover, they have been useful to
demonstrate the restriction of light-to-dark induced Ca
2+ fluxes to
chloroplasts only [8], and to investigate the integration of thylakoids in the Ca
2+ signaling network [9]. Nevertheless, an intrinsic
limitation of cell culture systems is given by their incompatibility
with systemic signaling studies, focused on the communication and
exchange of signals among different tissues and organs in the
elaboration of long-distance responses [10, 11].
Cell suspension cultures are also suitable experimental systems
to perform intracellular localization studies of recombinant proteins. Indeed, they can be obtained from transgenic plants expressing fluorescently tagged versions of the proteins of interest
[8]. Moreover, cell suspension cultures represent a convenient
system for the isolation of protoplasts (i.e., cells enzymatically
deprived of the cell wall), that can be isolated with high yields
starting from mid-exponential phase suspension-cultured cells.
Protoplasts can be useful for a number of studies, e.g., after transient expression of proteins of interest [12]. For example, we have
recently demonstrated that an Arabidopsis homolog of the mitochondrial calcium uniporter (cMCU) localizes to the chloroplast
envelope. The intracellular localization of this organellar Ca
2+ -permeable channel was ascertained by using Arabidopsis protoplasts
obtained by both photosynthetic and heterotrophic cell cultures,
transiently transformed with constructs encoding a GFP-fused version of cMCU and membrane markers of the different chloroplast
subcompartments [13].
It has to be noted that photosynthetic and heterotrophic cell
suspension cultures are somehow interconvertible, by modulating
sucrose concentration and light regime [5, 6, 8]. This is possible
thanks to the innate plasticity of plastids, highly dynamic organelles
that can interconvert in response to developmental and environmental cues [14, 15].
In this chapter, we describe in detail the protocols routinely
used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures. This simple
and precise guide will take the operator, including an absolute
beginner, through the basic steps of Arabidopsis cell cultures.
Modifications to the described protocols can be made and procedures can be tailored according to the host laboratory needs and
habits.
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Enrico Cortese et al.
and the advantages of working with Arabidopsis cell cultures containing functional chloroplasts have become increasingly evident
[5, 7].
Cell suspension cultures can be used for a number of structural
and functional studies, including signaling ones. The use of Arabidopsis photosynthetic and heterotrophic cell suspension cultures
has recently highlighted differences between non-green plastids
and chloroplasts in terms of organellar calcium signals in response
to environmental stimuli [8]. Moreover, they have been useful to
demonstrate the restriction of light-to-dark induced Ca
2+ fluxes to
chloroplasts only [8], and to investigate the integration of thylakoids in the Ca
2+ signaling network [9]. Nevertheless, an intrinsic
limitation of cell culture systems is given by their incompatibility
with systemic signaling studies, focused on the communication and
exchange of signals among different tissues and organs in the
elaboration of long-distance responses [10, 11].
Cell suspension cultures are also suitable experimental systems
to perform intracellular localization studies of recombinant proteins. Indeed, they can be obtained from transgenic plants expressing fluorescently tagged versions of the proteins of interest
[8]. Moreover, cell suspension cultures represent a convenient
system for the isolation of protoplasts (i.e., cells enzymatically
deprived of the cell wall), that can be isolated with high yields
starting from mid-exponential phase suspension-cultured cells.
Protoplasts can be useful for a number of studies, e.g., after transient expression of proteins of interest [12]. For example, we have
recently demonstrated that an Arabidopsis homolog of the mitochondrial calcium uniporter (cMCU) localizes to the chloroplast
envelope. The intracellular localization of this organellar Ca
2+ -permeable channel was ascertained by using Arabidopsis protoplasts
obtained by both photosynthetic and heterotrophic cell cultures,
transiently transformed with constructs encoding a GFP-fused version of cMCU and membrane markers of the different chloroplast
subcompartments [13].
It has to be noted that photosynthetic and heterotrophic cell
suspension cultures are somehow interconvertible, by modulating
sucrose concentration and light regime [5, 6, 8]. This is possible
thanks to the innate plasticity of plastids, highly dynamic organelles
that can interconvert in response to developmental and environmental cues [14, 15].
In this chapter, we describe in detail the protocols routinely
used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures. This simple
and precise guide will take the operator, including an absolute
beginner, through the basic steps of Arabidopsis cell cultures.
Modifications to the described protocols can be made and procedures can be tailored according to the host laboratory needs and
habits.
168
Enrico Cortese et al.