types and substructures can coexist in a same species [1, 6]. Studies
related with chromoplast differentiation have mainly focused on the
synthesis of carotenoids using biochemical and molecular
approaches [3, 5, 7]. In contrast, the biochemical, molecular and
cellular mechanisms underlying carotenoid sequestration and storage during chromoplast biogenesis remain largely unknown [1, 3].
During tomato fruit ripening, photosynthetically active chloroplasts differentiate into photosynthetically inactive chromoplasts
through a complex process distinguished by the breakdown of the
photosynthetic apparatus and a massive synthesis and deposition of
lycopene, the major carotenoid present in tomato fruit [8, 9]. The
structures found in a typical tomato fruit chromoplast include large
plastoglobules, an extensive laminar membranous system and long
osmiophilic filaments reported to accumulate lycopene in a crystalline form [10]. Plastoglobules are lipoprotein particles present in
plastids that have received an increasing interest during the last few
years after the discovery that they contain enzymes involved in the
synthesis of tocopherols and carotenoids [11]. Although chloroplast plastoglobules have been studied in detail, both at the structural and functional level, much less is known on chromoplast
plastoglobules [11]. The proteomic analysis of plastoglobules
isolated from red pepper chromoplasts has revealed the presence
of carotenogenic enzymes [12]. These results suggest an active role
of plastoglobules in carotenoid biosynthesis in addition to their
function in storage. The role of the other subplastidial structures
in carotenoid metabolism and storage during chromoplast differentiation in tomato fruit has not yet been characterized.
In recent years proteomics has become an efficient tool to study
the protein composition of chromoplasts as well as the dynamic
protein changes associated to their differentiation [13]. In tomato
fruit, chromoplast differentiation has mainly been associated with a
strong decrease in the abundance of proteins involved in photosynthesis and carbohydrate metabolism and an increase in the level of
carotenoid biosynthetic enzymes and stress-related proteins
[14]. In contrast, only a few proteins potentially involved in carotenoid sequestration and storage have been identified [15]. It is
likely that proteomic and metabolomic analysis targeting particular
chromoplast subfractions could provide new insights into biochemical and molecular processes related not only with carotenoid
sequestration and storage but also with the organization and regulation of carotenoid biosynthesis during tomato fruit ripening.
In the present protocol two procedures used for the isolation of
Arabidopsis plastoglobules [12, 16] have been adapted and merged
to fractionate tomato fruit chromoplasts isolated as described previously [17, 18]. The fractionation method described here allows
the isolation of fractions highly enriched in plastoglobules and
crystalline structures as well as other fraction containing different
membranous structures showing particular protein and metabolite
profiles.
190
Karel De Pourcq and Albert Boronat
related with chromoplast differentiation have mainly focused on the
synthesis of carotenoids using biochemical and molecular
approaches [3, 5, 7]. In contrast, the biochemical, molecular and
cellular mechanisms underlying carotenoid sequestration and storage during chromoplast biogenesis remain largely unknown [1, 3].
During tomato fruit ripening, photosynthetically active chloroplasts differentiate into photosynthetically inactive chromoplasts
through a complex process distinguished by the breakdown of the
photosynthetic apparatus and a massive synthesis and deposition of
lycopene, the major carotenoid present in tomato fruit [8, 9]. The
structures found in a typical tomato fruit chromoplast include large
plastoglobules, an extensive laminar membranous system and long
osmiophilic filaments reported to accumulate lycopene in a crystalline form [10]. Plastoglobules are lipoprotein particles present in
plastids that have received an increasing interest during the last few
years after the discovery that they contain enzymes involved in the
synthesis of tocopherols and carotenoids [11]. Although chloroplast plastoglobules have been studied in detail, both at the structural and functional level, much less is known on chromoplast
plastoglobules [11]. The proteomic analysis of plastoglobules
isolated from red pepper chromoplasts has revealed the presence
of carotenogenic enzymes [12]. These results suggest an active role
of plastoglobules in carotenoid biosynthesis in addition to their
function in storage. The role of the other subplastidial structures
in carotenoid metabolism and storage during chromoplast differentiation in tomato fruit has not yet been characterized.
In recent years proteomics has become an efficient tool to study
the protein composition of chromoplasts as well as the dynamic
protein changes associated to their differentiation [13]. In tomato
fruit, chromoplast differentiation has mainly been associated with a
strong decrease in the abundance of proteins involved in photosynthesis and carbohydrate metabolism and an increase in the level of
carotenoid biosynthetic enzymes and stress-related proteins
[14]. In contrast, only a few proteins potentially involved in carotenoid sequestration and storage have been identified [15]. It is
likely that proteomic and metabolomic analysis targeting particular
chromoplast subfractions could provide new insights into biochemical and molecular processes related not only with carotenoid
sequestration and storage but also with the organization and regulation of carotenoid biosynthesis during tomato fruit ripening.
In the present protocol two procedures used for the isolation of
Arabidopsis plastoglobules [12, 16] have been adapted and merged
to fractionate tomato fruit chromoplasts isolated as described previously [17, 18]. The fractionation method described here allows
the isolation of fractions highly enriched in plastoglobules and
crystalline structures as well as other fraction containing different
membranous structures showing particular protein and metabolite
profiles.
190
Karel De Pourcq and Albert Boronat