Download Subcellular Compartmentation of the Diterpene

Subcellular Compartmentation of the Diterpene Carnosic
Acid and Its Derivatives in the Leaves of Rosemary1
Sergi Munné-Bosch and Leonor Alegre*
Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645,
08028 Barcelona, Spain
The potent antioxidant properties of rosemary (Rosmarinus officinalis) extracts have been attributed to its major diterpene,
carnosic acid. Carnosic acid has received considerable attention in food science and biomedicine, but little is known about
its function in the plant in vivo. We recently found that highly oxidized diterpenes increase in rosemary plants exposed to
drought and high light stress as a result of the antioxidant activity of carnosic acid (S. Munné-Bosch, K. Schwarz, L. Alegre
[1999] Plant Physiol 121: 1047–1052). To elucidate the significance of the antioxidant function of carnosic acid in vivo we
measured the relative amounts of carnosic acid and its metabolites in different compartments of rosemary leaves. Subcellular
localization studies show that carnosic acid protects chloroplasts from oxidative stress in vivo by following a highly
regulated compartmentation of oxidation products. Carnosic acid scavenges free radicals within the chloroplasts, giving rise
to diterpene alcohols, mainly isorosmanol. This oxidation product is O-methylated within the chloroplasts, and the resulting
form, 11,12-di-O-methylisorosmanol, is transferred to the plasma membrane. This appears to represent a mechanism of a
way out for free radicals from chloroplasts. Carnosic acid also undergoes direct O-methylation within the chloroplasts, and
its derived product, 12-O-methylcarnosic acid, accumulates in the plasma membrane. O-methylated diterpenes do not
display antioxidant activity, but they may influence the stability of the plasma membrane. This study shows the relevance
of the compartmentation of carnosic acid metabolism to the protection of rosemary plants from oxidative stress in vivo.
Rosemary (Rosmarinus officinalis) leaf extracts show
a very high antioxidant activity and are increasingly
used as food additives, proposed as important human dietary factors, and investigated as inhibitors of
skin tumorgenesis (Singletary and Nelshoppen, 1991;
Schwarz et al., 1992; Huang et al., 1994). The main
compound responsible for the antioxidant activity is
the diterpene, carnosic acid (Aruoma et al., 1992),
which is the most abundant antioxidant found in the
leaves of rosemary. Carnosic acid is a lipophilic antioxidant that scavenges singlet oxygen, hydroxyl
radicals, and lipid peroxyl radicals, thus preventing
lipid peroxidation and disruption of biological membranes (Aruoma et al., 1992; Haraguchi et al., 1995).
Its radical scavenging activity follows a mechanism
analogous to that of other antioxidants such as
␣-tocopherol and is caused by the presence of two
O-phenolic hydroxyl groups found at C11 and C12 of
the molecule (Richheimer et al., 1999; Fig. 1).
Carnosic acid may give rise to carnosol after enzymatic dehydrogenation or to highly oxidized diterpenes such as rosmanol or isorosmanol after enzymatic dehydrogenation and free radical attack (Luis,
1991; Luis et al., 1994). Oxidative stress in vivo induced by drought or high light stress enhances the
formation of highly oxidized diterpenes due to the
1
This work was supported by the Programa Sectorial de Promoción General del Conocimiento (grant nos. DGICYT PB96 –1257
and BOS2000 – 0560).
* Corresponding author; e-mail [email protected]; fax
34 –93– 411–28 – 42.
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antioxidant activity of carnosic acid (Munné-Bosch et
al., 1999; Munné-Bosch and Alegre, 2000). In addition,
carnosic acid and isorosmanol can be O-methylated
to form 12-O-methylcarnosic acid and 11,12-di-Omethylisorosmanol, respectively. The methylation of
the O-phenolic hydroxyl groups eliminates the radical
scavenging activity of the molecule and increases its
lipid solubility (Brieskorn and Dömling, 1969).
The biological activity of a compound is determined
by its distribution within the plant cell. Diterpenes are
synthesized in plastids via a non-mevalonate isopentenyl diphosphate pathway (McGarvey and Croteau,
1995). In this pathway, isopentenyl diphosphate is
formed from pyruvate and glyceraldehyde 3phosphate, yielding 1-deoxy-d-xylulose-5-phosphate
(Kleinig, 1989; Lichtenthaler, 1999). The action of various phenyltransferases then generates geranylgeranyl
pyrophosphate (C20). This undergoes internal addition
to form copalyl pyrophosphate from which abietane
diterpenes such as carnosic acid are formed (McGarvey and Croteau, 1995). The formation of geranylgeranyl pyrophosphate and copalyl pyrophosphate has been localized in the plastids. However,
interaction between organelles may occur in the transfer of metabolites from plastids to sites of secondary
transformation such as endoplasmic reticulum-bound
Cyt P450 oxygenases, as it occurs in the synthesis of
gibberellins (a plant hormone) or abietic acid (a regulator of wound-induced responses in plants; McGarvey and Croteau, 1995). Carnosic acid may, therefore, occur in the chloroplasts or intracellular
membranes of rosemary leaves.
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Subcellular Localization of Diterpenes
11,12-di-O-methylisorosmanol was tested in different
tissues of rosemary (Fig. 2). The relative amounts of
diterpenes were similar in all the tissues tested, with
the concentration of carnosic acid 6-fold higher than
that of 12-O-methylcarnosic acid and that of carnosol,
which were found in similar amounts. Isorosmanol
was found at slightly lower concentrations than carnosol, 11,12-di-O-methylisorosmanol was approximately 10 times less abundant than isorosmanol, and
very small amounts of rosmanol were detected.
Diterpenes were not found in all the tissues tested.
The leaf was the tissue showing the highest concentrations of diterpenes. Diterpenes were also present
in the flowers of rosemary, although the concentrations found in the sepals were not comparable with
those found in the petals. Sepals contained approximately 30% fewer diterpenes than leaves, but 3.2
times more than petals. Diterpenes were also found
at low concentrations in seeds and trace amounts
were detected in stems. Roots did not contain
diterpenes.
Subcellular Fractionation
We recently showed that in conditions favoring
production of free radicals such as drought and high
light stress, the formation of highly oxidized diterpenes (rosmanol, isorosmanol, and 11, 12-di-O-methylisorosmanol) is enhanced in the leaves of rosemary
via the antioxidant activity of carnosic acid (MunnéBosch et al., 1999; Munné-Bosch and Alegre, 2000).
Thus, carnosic acid might play a major role in protecting the plant in vivo from oxidative stress. However, the significance of these results was not fully
clear, since studies on the subcellular localization of
this function were still lacking. In an attempt to characterize the in vivo function of carnosic acid this
study is aimed to elucidate the subcellular compartmentation of carnosic acid and its metabolites in the
leaves of rosemary.
Fractions enriched in chloroplasts, endoplasmic reticulum, Golgi apparatus, and plasma membrane
were prepared from fully developed young rosemary
leaves, which is the tissue containing the highest
concentrations of diterpenes. The identity and purity
of each fraction were determined by assaying
amounts and activities of appropriate markers (Table
I). Chloroplasts showed the highest amounts of chlorophylls and no or very little enzymatic activities that
are characteristic of other organelles. The identity
and purity of chloroplasts were also confirmed further by microscopic observation (data not shown).
The endoplasmic reticulum fraction showed the
highest activity of its marker enzyme NADPH-Cyt c
reductase, but no activity for the other enzymes
tested. The Golgi apparatus and the plasma membrane fractions showed the highest activity of their
marker enzymes, latent IDPase and vanadatesensitive ATPase, respectively, but little activity for
the other enzymes tested. All the fractions tested
showed very little activity of the mitochondrial enzyme marker Cyt c oxidase. Setting the specific
amount of markers or marker activities at 1 in the leaf
homogenate, the following relative enrichments in the
organelle fractions were obtained: chloroplast fraction,
10.1 for chlorophyll; endoplasmic reticulum fraction,
8.4 for NADPH-Cyt c reductase; Golgi apparatus fraction, 7.2 for latent IDPase; and plasma membrane fraction, 5.9 for vanadate-sensitive ATPase.
RESULTS
Subcellular Localization of Diterpenes
Figure 1. Diterpenes in rosemary leaves. The antioxidant activity of
diterpenes is given by the presence of two hydroxyl groups in ortho
position at C11 and C12. CA, Carnosic acid; MCA, 12-Omethylcarnosic acid; CAR, carnosol; ROS, rosmanol; ISO, isorosmanol; DMIR, 11,12-di-O-methylisorosmanol.
Distribution of Diterpenes in Different Tissues
The distribution of carnosic acid, 12-O-methylcarnosic acid, carnosol, rosmanol, isorosmanol, and
Plant Physiol. Vol. 125, 2001
The localization of diterpenes was studied using
subcellular fractions prepared from rosemary leaves.
The amount of carnosic acid, 12-O-methylcarnosic
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Munné-Bosch and Alegre
Figure 2. Tissue distribution of the diterpenes
carnosic acid (CA), 12-O-methylcarnosic acid
(MCA), carnosol (CAR), rosmanol (ROS), isorosmanol (ISO), and 11,12-di-O-methylisorosmanol (DMIR) in rosemary plants. Results, given
in milligrams per gram of dry weight, correspond to the means ⫾ SE of triplicate experiments. Sd, Seed; Rt, root; St, stem; Le, leaf; Se,
sepal; Pe, petal. aND, Not detected; bTr, trace.
acid, carnosol, rosmanol, isorosmanol, and 11,12-diO-methylisorosmanol was determined in chloroplasts, endoplasmic reticulum, Golgi apparatus, and
plasma membrane fractions (Fig. 3). Carnosic acid
was only found in the chloroplasts of rosemary
leaves, along with carnosol, rosmanol, and isorosmanol. In contrast, the O-methylated forms of carnosic
acid and isorosmanol, 12-O-methylcarnosic acid, and
11,12-di-O-methylisorosmanol, respectively, were
found in chloroplasts, endoplasmic reticulum, Golgi
apparatus, and plasma membrane fractions. The relative amounts of carnosic acid, carnosol, rosmanol,
and isorosmanol found in the chloroplasts were similar to those found in the leaves (Fig. 2); carnosic acid
was the most abundant diterpene, followed by carnosol, isoromanol, and rosmanol. O-methylated
diterpenes accumulated in the plasma membrane.
The amount of 12-O-methylcarnosic acid found in the
plasma membrane was 2.8-fold higher than that
found in the Golgi apparatus, 3.6-fold higher than
that found in the endoplasmic reticulum, and 5-fold
higher than that found in the chloroplasts. The
amount of 11,12-di-O-methylisorosmanol found in
the plasma membrane was 6-fold higher than that
Table I. Distribution of marker compounds and marker enzyme activities in subcellular fractions of rosemary leaves
Data are shown in absolute values and as a percentage of the maximum for each marker (in brackets). Results correspond to the mean ⫾
of triplicate experiments.
Marker
a
Chlorophyll
NADPH-Cyt c reductaseb
Latent IDPased
Vanadate-sensitive ATPased
Cyt c oxidased
Chloroplast
Endoplasmic Reticulum
185.2 ⫾ 1.0 (100)
ND c (0)
1.8 ⫾ 0.4 (⬍1)
ND (0)
2.6 ⫾ 0.65
0 (0)
38.9 ⫾ 0.7 (100)
ND (0)
ND (0)
2.2 ⫾ 0.2
mg (chlorophyll a ⫹ b) g⫺1 protein.
protein.
a
1096
b
nmol Cyt c min⫺1 mg⫺1 protein.
Golgi Apparatus
Plasma Membrane
0 (0)
11.5 ⫾ 1.0 (30)
153.9 ⫾ 5.7 (100)
1.4 (⬍1)
0.7 ⫾ 0.5
c
ND, Not detected.
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d
SE
0 (0)
7.5 ⫾ 2.8 (19)
33.8 ⫾ 5.6 (22)
295.8 ⫾ 60.6 (100)
0.8 ⫾ 0.1
␮mol phosphate min⫺1 mg⫺1
Plant Physiol. Vol. 125, 2001
Subcellular Localization of Diterpenes
Figure 3. Subcellular distribution of the diterpenes carnosic acid (CA), 12-O-methylcarnosic
acid (MCA), carnosol (CAR), rosmanol (ROS),
isorosmanol (ISO), and 11,12-di-O-methylisorosmanol (DMIR) in the leaves of rosemary. Results,
given in milligrams per gram of dry weight,
correspond to the means ⫾ SE of triplicate experiments. Chl, Chloroplast; ER, endoplasmic
reticulum; GA, Golgi apparatus; PM, plasma
membrane. aND, Not detected.
found in the Golgi apparatus, 8.3-fold higher than
that found in the endoplasmic reticulum, and 16.6-fold
higher than that found in the chloroplasts (Fig. 3).
Setting the specific amount of diterpenes at 1 in the
leaf homogenate, the following relative enrichments in
the organelle fractions were obtained: chloroplast fraction, 5.3 for carnosic acid, 4.0 for 12-O-methylcarnosic
acid, 5.3 for carnosol, 4.8 for rosmanol, 4.2 for isorosmanol, and 4.0 for 11,12-di-O-methylisorosmanol; endoplasmic reticulum fraction, 5.5 for 12-O-methylcarnosic acid and 8 for 11,12-di-O-methylisorosmanol;
Golgi apparatus fraction, 7.3 for 12-O-methylcarnosic
acid and 10.5 for 11,12-di-O-methylisorosmanol; and
plasma membrane fraction, 18.3 for 12-O-methylcarnosic acid and 63.3 for 11,12-di-O-methylisorosmanol.
Occurrence of Diterpene Esters and Glycosides
The possible occurrence of diterpene esters or glycosides in the leaves of rosemary was also tested (Fig.
4). If conjugates were present, the treatment with HCl
or KOH might cause a rupture of the bond of the
conjugated form resulting in an increase of free diterpenes. However, the acid and alkali treatments resulted in a decrease in the concentration of diterpePlant Physiol. Vol. 125, 2001
nes rather than an increase. This decrease was similar
to that observed for pure carnosic acid in solution.
Thus, the presence of diterpene conjugates, at least in
significant amounts, may be excluded.
DISCUSSION
Diterpenes play diverse functional roles in plants,
acting as hormones (gibberellins), regulators of woundinduced responses (abietic acid), photosynthetic pigments (the phytyl chain of chlorophylls), and antioxidants (the phytyl moiety of tocopherols; McGarvey and
Croteau, 1995; Lichtenthaler et al., 1997). Although tocopherols and carotenoids are, among lipid-soluble antioxidants, the best-characterized groups of compounds
in their function of protecting the plant from oxidative
stress, plants contain other compounds (i.e. flavonoids
and diterpenes) displaying high antioxidant properties
(Schwarz and Ternes, 1992; Rice-Evans et al., 1997). In
vivo studies have shown that the diterpene carnosic
acid may protect biological membranes from oxidative
damage (Haraguchi et al., 1995), and that under
drought- and high light-induced oxidative stress
conditions, the amounts of highly oxidized diterpenes (i.e. rosmanol, isorosmanol, and 11, 12-di-O-
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Munné-Bosch and Alegre
Figure 4. Influence of acid and alkali treatments on the extraction of
carnosic acid (CA) from rosemary leaves. The extraction of diterpenes in methanol (Me) was compared with their extraction in the
presence of HCl (⫹ HCl) and KOH (⫹ KOH) to study the possible
occurrence of diterpenoid esters or glycosides. A control experiment
using pure carnosic acid in solution was performed. Similar changes
to those obtained for carnosic acid were also observed with the other
diterpenes. Results are given in absolute values (milligrams per gram
of dry weight or mililiter, bars) and as a percentage of the initial
values (%, black symbols). Results correspond to the means ⫾ SE of
three independent replicates.
methylisorosmanol) in rosemary leaves increase due to
the antioxidant activity of carnosic acid (Munné-Bosch
et al., 1999; Munné-Bosch and Alegre, 2000). This study
shows the subcellular compartmentation of carnosic
acid and its oxidation products in rosemary leaves (Fig.
5). This allows us to fully explain previous results and
better characterize the in vivo function of carnosic acid
in rosemary plants.
Carnosic acid was only found in chloroplasts. Copalyl pyrophosphate, the immediate precursor of the
diterpene carnosic acid, has also been found in these
organelles (McGarvey and Croteau, 1995), thus suggesting that carnosic acid is synthesized in the chloroplasts. The oxidation products of carnosic acid,
rosmanol, and isorosmanol were also only found in
the chloroplasts, thus indicating that carnosic acid
functions as an antioxidant in the chloroplasts of
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rosemary leaves. This demonstrates that the antioxidant function of carnosic acid is linked to the chloroplasts, which are the organelles most exposed to
oxidative damage in plant cells. Chloroplasts are organelles particularly liable to oxygen toxicity, since
they function under high oxygen tensions and in the
light. As a result of a stress-induced de-regulation of
the photosynthetic activity of chloroplasts, more free
radicals (singlet oxygen, superoxide anion, hydroxyl
radicals, and peroxyl radicals) may be generated in
this organelle, which may cause disruption of membranes and cell death (Foyer et al., 1994; Osmond et
al., 1997; Asada, 1999). Environmental constraints
such as drought and high light stress may inhibit the
normal functioning of the photosynthetic apparatus
and cause oxidative damage to the chloroplasts if the
plants are not protected by antioxidant defenses
(Björkman, 1987; Smirnoff, 1993). Therefore, the compartmentation of the antioxidant function of carnosic
acid in chloroplasts might be an adaptive mechanism
that rosemary plants have evolved to withstand
drought and high light stress typical of the Mediterranean climate.
The lipophilic nature of carnosic acid and its antioxidant properties suggest that carnosic acid may
play a role similar to ␣-tocopherol in scavenging free
radicals formed as a result of the photosynthetic
activity of chloroplasts. As occurs with ␣-tocopherol
(Fryer, 1992; Shintani and DellaPenna, 1998), diterpenes are found at their highest concentrations in photosynthetic tissues (i.e. leaves and sepals), and appreciable amounts of these compounds are also found in
seeds and petals. The leaves of rosemary contain
amounts of ␣-tocopherol comparable with other species (Munné-Bosch and Alegre, 2000), which indicates that this species is not deficient in this antioxidant. Thus, carnosic acid may cooperate with
␣-tocopherol in chloroplasts, rather than replace its
activity. Some authors have suggested that carnosic
acid could interact synergically with ␣-tocopherol by
reducing the tocopheryl radicals to active ␣-tocopherol (Hopia et al., 1996). This might explain the
great tolerance of rosemary to drought and high light
stress, but such an interaction needs to be examined
in vivo.
The results also indicate that the oxidation product
of carnosic acid, isorosmanol, is O-methylated within
the chloroplasts and that the derived diterpene,
11,12-di-O-methylisorosmanol, is transferred from
the chloroplasts to the plasma membrane. Although
we did not find 11,12-di-O-methylrosmanol, the
O-methylated form of rosmanol, it is likely to occur
in rosemary leaves, as it has been described in other
species (Luis et al., 1994). Thus, the mechanism accounting for isorosmanol may also apply for its isomer, rosmanol. The formation of O-methylated diterpenes from carnosic acid oxidation products within
the chloroplasts may represent, therefore, a mechanism of a way out of free radicals from chloroplasts.
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Plant Physiol. Vol. 125, 2001
Subcellular Localization of Diterpenes
Figure 5. Subcellular compartmentation of carnosic acid and its metabolites in the leaves of
rosemary. CA, Carnosic acid; CAR, carnosol;
MCA, 12-O-methylcarnosic acid; ROS, rosmanol; ISO, isorosmanol; DMIR, 11,12-di-O-methylisorosmanol; frs, free radical scavenging.
The accumulation of O-methylated diterpenes in the
plasma membrane indicates that these compounds
may have a function apart from being antioxidants,
since the O-methylation of one of two hydroxyl
groups found at C11 and C12 leads to a decrease in the
free radical scavenging activity of the molecule (Brieskorn and Dömling, 1969). O-methylated diterpenes accumulating in the plasma membrane may have a
similar function to sterols. Although O-methylated
diterpenes do not display antioxidant activity, they
retain at least one hydroxyl group in the molecule.
This hydroxyl group may form hydrogen bonds with
the head group of a phospholipid and its dimensions
may allow cooperative van der Waals attractive
forces to reinforce and stabilize the lipid chain (Havaux, 1998). As occurs with sterols, the amount of
O-methylated diterpenes accumulated in the plasma
membrane may affect the integrity of the cells and
influence plant growth and development (Schaller et
al., 1998; Brown and Goldstein, 1999). The same
may apply for diterpenes found in the chloroplasts.
It is well known that the lipid-soluble antioxidants
␣-tocopherol and ␤-carotene may significantly influence the stability of membranes in chloroplasts (Havaux, 1998). The leaves of rosemary contain 100 times
more carnosic acid than ␣-tocopherol and 35 times
more carnosic acid than ␤-carotene (Munné-Bosch
and Alegre, 2000). Thus, carnosic acid is thought to
affect the stability of membranes in the chloroplasts
more than these lipid-soluble antioxidants.
We conclude that carnosic acid metabolism plays a
dual role in rosemary plants. First, the localization of
the antioxidant function of carnosic acid in the chloroplasts and the enhanced formation of oxidation
products in stress conditions indicates that carnosic
acid protects rosemary plants from environmental
constraints by scavenging free radicals within the
chloroplasts. Second, the large amounts of antioxidant
diterpenes and nonantioxidant diterpenes found in
Plant Physiol. Vol. 125, 2001
chloroplasts and intracellular membranes, respectively, suggest that carnosic acid metabolism may also
play a role in the stability of cell membranes.
MATERIALS AND METHODS
Plant Material
Rosemary (Rosmarinus officinalis) plants grown at the
experimental fields of the University of Barcelona were
used for this study. Six plants of the same genetic origin,
age (2 years old), and height (1 m) were chosen. Diterpenes
were determined in plant tissues (i.e. roots, stems, leaves,
sepals, and petals) and subcellular fractions of leaves (i.e.
chloroplasts, endoplasmic reticulum, Golgi apparatus, and
plasma membrane) from material collected at predawn
during January and February 2000. Diterpenes were also
determined in seeds obtained from Semillas Fitó (Barcelona). For the determination of diterpenes in different tissues, the plant material was collected, immediately frozen
in liquid nitrogen, and freeze-dried prior to extraction. For
the determination of diterpenes in subcellular fractions, the
leaves were collected and cells were immediately fractionated as described below. The subcellular fractions obtained
were immediately frozen in liquid nitrogen and freezedried prior to extraction.
Isolation of Chloroplasts
Chloroplasts were isolated by an adaptation of the
method of Walker and Weinstein (1991). The abaxial epidermis of leaves was mechanically removed with the help
of a scalpel to avoid the interference of trichomes in the
isolation procedure. Leaf tissue (5 g) was ground with an
ice-chilled mortar and pestle with a 20-mL isolation buffer,
pH 7.8, containing 0.5 m sorbitol, 50 mm Tricine, 1 mm
dithiothreitol, 1 mm MgCl2, 1 mm butylated hydroxytoluene, and 0.1% (w/v) bovine serum albumin (BSA). The
homogenate was filtered through four layers of cheesecloth
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Munné-Bosch and Alegre
and centrifuged at 2,500g for 4 min. The pellet was resuspended in 10 mL of isolation buffer and then centrifuged at
200g for 1 min. The chloroplasts in the supernatant were
sedimented by centrifugation at 2,500g for 4 min. Chloroplasts were purified by resuspending the pellets in 2 mL of
isolation buffer, layering onto 10 mL of 25% (v/v) Percoll
(in isolation buffer), and centrifuging at 15,800g for 20 min.
The chloroplasts pellets were resuspended in 10 mL of
isolation buffer lacking BSA, centrifuged at 2,500g for 4
min, and used immediately. The whole procedure was
carried out in dimmed room light at 4°C.
Isolation of Endoplasmic Reticulum, Golgi
Apparatus, and Plasma Membrane Fractions
A Suc-density gradient and a polymer two-phase system
were used for the isolation of plasma and intracellular
membranes (Briskin et al., 1987; Moreau et al., 1998). Leaf
tissue (5 g) was homogenized at high speed for 60 s with a
Polytron probe (Kinematica, Luzern, Switzerland) in 20 mL
of buffer consisting of 10 mm KH2PO4, pH 8.2, with 0.5 m
sorbitol, 5% (w/v) polyvinylpyrrolidone (40,000), 0.5%
(w/v) BSA, 2 mm salicylhydroxamic acid, 1 mm butylated
hydroxytoluene, and 1 mm phenylmethylsulfonyl fluoride.
The homogenate was filtered through six layers of cheesecloth and was centrifuged at 1,000g for 10 min, 10,000g for
10 min, and 150,000g for 60 min. The resulting microsomal
pellet was resuspended in 10 mL of buffer consisting of 10
mm KH2PO4, pH 8.2, with 0.5 m sorbitol. One-half of the
suspension was loaded onto a discontinuous Suc-density
gradient consisting of 2.5 mL of 37% (w/v) Suc, 3.5 mL of
25% (w/v) Suc, and 3.5 mL of 18% (w/v) Suc. After centrifuging at 80,000g for 150 min, membranes at the 18%/
25% (endoplasmic reticulum fraction) and 25%/37% (Golgi
apparatus fraction) Suc interface were collected, diluted
with phosphate buffer, pH 8.2, centrifuged at 100,000g for
60 min, and used immediately. The other one-half of the
microsomal suspension was mixed with a polymer (polyethylene glycol [PEG] 4000/Dextran T500 mixture) in 0.5 m
sorbitol containing 10 mm KH2PO4 and 40 mm NaCl, pH
7.8, to obtain final PEG and Dextran concentrations of 6%
(w/w). The solution (final volume of 28 mL) was centrifuged for 15 min at 1,000g and the PEG-enriched upper
phase (12 mL) was recovered without disturbing the interface. The upper phase was repartitioned twice to produce a
chlorophyll-free preparation. Plasma membranes were recovered after centrifuging at 150,000g for 60 min, and were
used immediately. The whole procedure was carried out in
dimmed room light at 4°C.
Assays of Markers for Subcellular Fractions
Specific subcellular compartments were identified by
assays for the following markers: chloroplasts, chlorophyll;
endoplasmic reticulum, NADPH-Cyt c reductase; Golgi
apparatus, latent IDPase; and plasma membrane,
vanadate-sensitive ATPase. Marker assays were performed
immediately after isolation of the corresponding fraction.
Chlorophylls were measured spectrophotometrically in
1100
80% (v/v) acetone extracts (Lichtenthaler and Wellburn,
1983). The NADPH-Cyt c reductase assay was performed at
25°C in a 3-mL reaction volume containing 0.1 mL of
membrane suspension (10 ␮g of protein), 0.1 mL of 50 mm
NaCN, 0.2 mL of 0.45 mm Cyt c, and 2.5 mL of 50 mm
sodium phosphate buffer, pH 7.5. The reaction was started
by the addition of 0.1 mL of 3 mm NADPH, and the
reduction of Cyt c was followed spectrophotometrically as
an absorbance increase at 550 nm (Lord, 1987). Latent
IDPase was assayed in a 3-mL reaction volume containing
0.1 mL of membrane suspension (10 ␮g of protein), 0.1 mL
of 3 mm MgSO4, 0.1 mL of 50 mm KCl, and 2.6 mL of 30 mm
Tris-MES [2-(N-morpholino)-ethanesulfonic acid] buffer
(MES titrated with Tris to pH 7.5). The reaction was started
by the addition of 0.1 mL of 3 mm IDP (sodium salt), and
the released Pi was determined on freshly isolated membranes and after 6 d of storage at 2°C to 4°C by using
ammonium molybdate (Briskin et al., 1987). Vanadatesensitive ATPase was assayed in a 3-mL reaction volume
containing 0.1 mL of membrane suspension (10 ␮g of protein), 0.1 mL of 3 mm MgSO4, 0.1 mL of 50 mm KCl, and 2.5
mL of 30 mm Tris-MES buffer (MES titrated with Tris to pH
6.5), in the presence or absence of 0.1 mL of 50 ␮m Na3VO4.
The reaction was started by the addition of 0.1 mL of 3 mm
ATP. The ATP substrate was present as Tris salt after
treatment with Dowex 50-W exchange resin (H⫹ form). The
assay was performed at 38°C for 30 min, and the released
Pi was determined by using ammonium molybdate
(Briskin et al., 1987; Fan et al., 1999). The specific activity of
Cyt c oxidase (a mitochondria marker enzyme) was assayed at 25°C in a 3-mL reaction volume containing 0.1 mL
of membrane suspension (10 ␮g of protein), 0.1 mL of 0.3%
(w/v) Triton X-100, and 2.7 mL of 50 mm sodium phosphate buffer, pH 7.5. The reaction was started by the addition of 0.1 mL of 0.45 mm dithionite-reduced Cyt c, and
the decrease at A550 was monitored spectrophotometrically
(Fan et al., 1999). Protein concentration was determined by
the method of Bradford (1976) using a kit (Bio-Rad, Hercules, CA) with BSA as a standard.
Control Experiments
To test the possible re-distribution of diterpenes between
different subcellular organelles during subcellular fractionation, the following experiment was performed. Carnosic
acid (90% purity, 5 mg) was added to the initial homogenate. After isolation of the corresponding subcellular fractions, the amount of diterpenes was determined. The
amounts of diterpenes obtained in chloroplast-, endoplasmic reticulum-, Golgi apparatus-, and plasma membraneenriched fractions were not significantly different (P ⱕ
0.05, ANOVA) from those obtained when carnosic acid was
not added to the homogenate.
Hydrolisis of Esters and Glycosides
To investigate the presence of diterpenoid esters or glycosides, freeze-dried leaves were hydrolized by heating at
60°C for 1 h with 5 mL of 6% (w/v) KOH in methanol or
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Plant Physiol. Vol. 125, 2001
Subcellular Localization of Diterpenes
with 5 mL of 20% (v/v) 6 m HCl in methanol (Hartmann
and Benveniste, 1987; Hertog et al., 1992). Controls were
carried out by heating the leaves at 60°C for 1 h with 5 mL
of methanol and by testing the degradation of carnosic acid
(90% purity) under the same conditions.
Determination of Diterpenes
For the determination of diterpenes, the corresponding
samples were freeze-dried and immediately analyzed.
Diterpenes were determined as described (Munné-Bosch
et al., 1999). In short, the samples were extracted with
methanol containing 0.005% (w/w) citric acid and 0.005%
(w/w) isoascorbic acid, and were sonicated for 20 s
(Vibra-Cell Ultrasonic Processor, Sonics and Materials,
Danbury, CT). Diterpenes were separated on an octadecyl
(C18) silica Hypersil 5-␮m column (250 ⫻ 4 mm, Teknokroma, St. Cugat, Spain) during 52 min at a flow rate of
0.6 mL min⫺1. The eluants consisted of A, 51% (w/v)
acetonitrile and 49% (w/v) water, containing 0.83% (w/v)
2 m citric acid; and B, 97% (w/v) acetonitrile and 3%
(w/v) water, containing 0.5% (w/v) 2 m citric acid. The
following gradient was used: 0 to 20 min, 100% A and 0%
B; 20 to 34 min decreasing to 50% A and 50% B; 34 to 40
min decreasing to 0% A and 100% B; 40 to 48 min, increasing to 100% A and 0% B; and 48 to 52 min, 100% A and 0%
B. Individual diterpenes were identified by their characteristic mass spectra. Carnosic acid (98% purity) was used
for calibration. All diterpenes were quantified relative to
carnosic acid at 230 nm (Spectralphotometer 430 Kontron,
Zurich), because the UV spectra of other diterpenes are
similar to that of carnosic acid.
ACKNOWLEDGMENTS
We thank the Serveis Cientı́fico-tècnics from the University of Barcelona for technical assistance and Dr. Karin
Schwarz (University of Kiel) for kindly providing us with
carnosic acid. We also thank the University of Barcelona for
the grant given to S.M.
Received June 19, 2000; returned for revision September 5,
2000; accepted October 22, 2000.
LITERATURE CITED
Aruoma OI, Halliwell B, Aeschbach R, Löliger J (1992)
Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid. Xenobiotica 22: 257–268
Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50: 601–639
Björkman O (1987) Responses to different quantum flux
density. In J Biggings, ed, Progress in Photosynthesis
Research. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp 11–18
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
Plant Physiol. Vol. 125, 2001
the principle of protein-dye binding. Anal Biochem 72:
248–254
Brieskorn CH, Dömling H (1969) Carnosolsäure, der wichtige antioxidativ wirksame Inhaltsstoff des Rosmarinund Salbeiblattes. Z Lebens Unter Fors 141: 10–16
Briskin DP, Leonard RT, Hodges TK (1987) Isolation of
the plasma membrane: membrane markers and general
principles. Methods Enzymol 148: 543–558
Brown MS, Goldstein JS (1999) A proteolytic pathway that
controls the cholesterol content of membranes, cells, and
blood. Proc Natl Acad Sci USA 96: 11041–11048
Fan L, Zheng S, Cui D, Wang X (1999) Subcellular distribution and tissue expression of phospholipase D␣, D␤,
and D␥ in Arabidopsis. Plant Physiol 119: 1371–1378
Foyer CH, Descourvières P, Kunert KJ (1994) Protection
against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant Cell Environ 17:
507–523
Fryer MJ (1992) The antioxidant effects of thylakoid vitamin E (␣-tocopherol). Plant Cell Environ 15: 381–392
Haraguchi H, Saito T, Okamura N, Yagi A (1995) Inhibition of lipid peroxidation and superoxide generation by
diterpenoids from Rosmarinus officinalis. Planta Med 61:
333–336
Hartmann MA, Benveniste P (1987) Plant membrane sterols: isolation, identification and biosynthesis. Methods
Enzymol 148: 632–650
Havaux M (1998) Carotenoids as membrane stabilizers in
chloroplasts. Trends Plant Sci 3: 147–151
Hertog MGL, Hollman PCH, Venema DP (1992) Optimization of a quantitative HPLC determination of potentially anticarcinogenic flavonoids in vegetables and
fruits. J Agri Food Chem 40: 1591–1598
Hopia AI, Huang S, Schwarz K, German JB, Frankel EN
(1996) Effect of different lipid systems on antioxidant
activity of rosemary constituents carnosol and carnosic
acid with and without ␣-tocopherol. J Agri Food Chem
44: 2030–2036
Huang M, Ho C, Wang ZY, Ferraro T, Lou Y, Stauber K,
Ma W, Georgiadis C, Laskin JD, Conney AH (1994)
Inhibition of skin tumorgenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res 54:
701–708
Kleinig H (1989) The role of plastids in isoprenoid biosynthesis. Annu Rev Plant Physiol Plant Mol Biol 40: 39–59
Lichtenthaler HK (1999) The 1-deoxy-d-xylulose-5phosphate pathway of isoprenoid biosynthesis in plants.
Annu Rev Plant Physiol Plant Mol Biol 50: 47–65
Lichtenthaler HK, Schwender J, Disch A, Rohmer M
(1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Lett 400: 271–274
Lichtenthaler HK, Wellburn AR (1983) Determination of
total carotenoids and chlorophylls a and b of leaf extracts
in different solvents. Biochem Soc Trans 11: 591–592
Lord JM (1987) Isolation of endoplasmic reticulum: general
principles, enzymatic markers, and endoplasmic
reticulum-bound polysomes. Methods Enzymol 148:
576–584
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1101
Munné-Bosch and Alegre
Luis JG (1991) Chemistry, biogenesis, and chemotaxonomy
of the diterpenoids of Salvia. In JB Harborne, FA TomasBarberan, eds, Ecological Chemistry and Biochemistry of
Plant Terpenoids. Clarendon Press, Oxford, pp 63–82
Luis JG, Quiñones W, Grillo TA, Kishi MP (1994) Diterpenes from the aerial part of Salvia columbariae. Phytochemistry 35: 1373–1374
McGarvey DJ, Croteau R (1995) Terpenoid metabolism.
Plant Cell 7: 1015–1026
Moreau P, Hartmann M, Perret M, Sturbois-Balcerak B,
Cassagne C (1998) Transport of sterols to the plasma
membrane of leek seedlings. Plant Physiol 117: 931–937
Munné-Bosch S, Alegre L (2000) Changes in carotenoids,
tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in
Rosmarinus officinalis plants. Planta 210: 925–931
Munné-Bosch S, Schwarz K, Alegre L (1999) Enhanced
formation of ␣-tocopherol and highly oxidized abietane
diterpenes in water-stressed rosemary plants. Plant
Physiol 121: 1047–1052
Osmond B, Badger M, Maxwell K, Björkman O, Leegod R
(1997)) Too many photons: photorespiration, photoinhibition and photooxidation. Trends Plant Sci 2: 119–120
Rice-Evans CA, Miller NJ, Paganga G (1997) Antioxidant
properties of phenolic compounds. Trends Plant Sci 2:
152–159
Richheimer SL, Bailey DT, Bernart MW, Kent M, Vininski JV, Anderson LD (1999) Antioxidant activity and
oxidative degradation of phenolic compounds isolated
from rosemary. Recent Res Dev Oil Chem 3: 45–58
1102
Schaller H, Bouvier-Navé P, Benveniste P (1998) Overexpression of an Arabidopsis cDNA encoding a sterol-C241methyltransferase in tobacco modifies the ratio of 24methyl cholesterol to sitosterol and is associated with
growth reduction. Plant Physiol 118: 461–469
Schwarz K, Ternes W (1992) Antioxidative constituents of
Rosmarinus officinalis and Salvia officinalis: I. Isolation of
carnosic acid and formation of other phenolic diterpenes.
Z Lebens Unter Fors 195: 99–103
Schwarz K, Ternes W, Schmauderer E (1992) Antioxidative constituents of Rosmarinus officinalis and Salvia officinalis: III. Stability of phenolic diterpenes of rosemary
extracts under thermal stress as required for technological processes. Z Lebens Unter Fors 195: 104–107
Shintani D, DellaPenna D (1998) Elevating the vitamin E
content of plants through metabolic engineering. Science
282: 2098–2100
Singletary KW, Nelshoppen JM (1991) Inhibition of 7,12dimethylbenzanthracene- (DMBA) induced mammary
tumorgenesis and of in vivo formation of mammary
DMBA-DNA adducts by rosemary extract. Cancer Lett
60: 169–175
Smirnoff N (1993) Tansley review no. 52: the role of active
oxygen in the response of plants to water deficit and
desiccation. New Phytol 125: 27–58
Walker CJ, Weinstein JD (1991) In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of
the activity into soluble and membrane-bound fractions
Proc Natl Acad Sci USA 88: 5789–5793
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
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