Sitosterol (1) is usually the major

Sitosterol (1) is usually the major sterol found in plant tissues,
and an increased sitosterol (1) to stigmasterol (2) ratio has been
suggested as a mechanism of plant adaptation to stress (Douglas,
1985). This, was confirmed in our study where the proportion of
sitosterol (1) to stigmasterol (2) was about 7-fold higher in the
young leaves of plants exposed to low UV-B irradiance. When their
levels were compared in control plants no differences were
observed between young and mature leaf tissues, and only under
UV-B treatment an increase was in these compounds detected. In
previous work, with grapevine mature leaves, the sitosterol (1)
content was 5-fold higher than that of stigmasterol (2) (Berli et
al., 2010). However, the ratio of sitosterol (1) to stigmasterol (2)
could be influenced by the plant species, stress intensity (Douglas,
1985) or tissue ontogeny (Geuns, 1973). Additionally, levels of the
triterpene lupeol (3) increased under low UV-B. However in this
case, such an increase was more notable in mature leaves, suggesting
metabolism from an immediate precursor and not de novo synthesis
via isoprenoid metabolism. Squalene synthase (SS) catalyses
the first step of the isoprenoid pathway towards sterol and triterpene
biosynthesis (Abe et al., 1993). In plants, both sterols and
triterpenes are synthesized as products of cyclization of 2,3-oxidosqualene
(14), a reaction catalyzed by oxidosqualene cyclases
(OSCs; see Fig. 8). It has been suggested that biosynthesis of
triterpenes occurs when sterol formation has been sacrificed
(Flores-Sánchez et al., 2002; Kamisako et al., 1984). However,
simultaneous enhancement of sterols and triterpene biosynthesis
has been reported (Han et al., 2010; Lee et al., 2004). Probably,
overproduction of squalene (13) in UV-B irradiated plants stimulates
both, phytosterol and triterpene biosynthesis. In any case,
lupeol (3) and sterols, in addition to regulate fluidity and permeability
of the membranes have demonstrated capacity to intercept
free radicals (Bracco et al., 1981; Hartmann, 1998; Saleem et al.,
2001). Although the biological function of lupeol (3) in plants is
less clear, it may have protective functions. It has been shown in
mammal tissues that lupane derivatives have antioxidant, antiinflammatory
and antiviral properties (Lee et al., 2007; Saleem
et al., 2004, 2005a,b, 2008). Other studies indicate that lupeol (3)
also plays a role in stabilization of lipids (Saratha et al., 2011)
and changes the tissue redox balance by scavenging free radicals (Nagaraj et al., 2000; Sunitha et al., 2001). Thus, these triterpenes
may protect leaf membranes against potential photo-oxidative
damage generated by low UV-B as part of a mechanism for grape
acclimation to UV-B radiation. When the UV-B dose was administrated
at low fluence rate, the results suggested de novo synthesis
of membrane-related sterols. In fact, the highest levels of sitosterol
(1) and stigmasterol (2) were found in young leaves in correlation
with high TPS activity. This type of reaction is consistent with an
adaptative response that prepares tissues to cope with adverse
environmental conditions. Notwithstanding, in previous experiments
(Berli et al., 2010) it was found that such structural resistance
throughout an enhancement of membrane sterols is more
associated with ABA (11) effects than a direct consequence of
UV-B action. Therefore, as ABA (11) levels were also increased by
the low UV-B treatment, such levels may account for increase of
membrane-sterols in young tissues.
On the other hand, UV-B treatment somewhat promoted synthesis
of a-tocopherol (5), c-tocopherol (4) and phytol (6), all diterpenes
derived of the plastidic methylerythritol phosphate (MEP)
pathway (Lichtenthaler et al., 1997). These antioxidant compounds
prevent photo-oxidative deterioration of unsaturated fatty acids,
lipids and lipoproteins in the cell membrane of plants by detoxification
of ROS (Fahrenholtz et al., 1974; Neely et al., 1988), but are also
involved in the modulation of membrane fluidity and permeability
(Fryer, 1992). It is well known that tocopherols can scavenge ROS
directly by reactions through hydroxyl radicals avoiding propagation
of lipid peroxidation in thylakoid membranes, and forming a
first line of defense against membrane photo-degradation (Asada,
1994; Foyer et al., 1994). Therefore, they are essentials components
for the plant protection against environmental stress and light-induced
disorders (reviewed by Fryer, 1992). In our experiments,
these diterpenes were even more abundant in mature leaves and
when the grapevine plants were irradiated with high UV-B. The
antioxidant function of tocopherols may contribute to avoid irreversible
damages of membranes keeping them functional after
the UV-B treatment (Munné-Bosch and Alegre, 2000). Experimental
evidence has shown that tocopherols confer a protective effect to
the photosynthetic apparatus under thermal and oxidative stress
conditions (Copolovici et al., 2005; Delfine et al., 2000), acting as antioxidants that protect the membranes against peroxidation and
ROS (Loreto and Velikova, 2001; Loreto et al., 2001). Thus, the
induction and accumulation of both tocopherols (4, 5) and phytol
(6) could be interpreted as a mechanism to shorten duration of
the ‘‘oxidative burst’’ and to protect grapevine tissues against high
UV-B induced-ROS. These results are in agreement with the expression
of genes involved in different antioxidant defense systems promoted
by exposure of grape plants to high UV-B, such as genes
encoding several heat shock proteins (with mitochondrial, chloroplastic,
and cytoplasmic localization), different kind of lectins, and
antioxidant enzymes (Pontin et al., 2010).
Among the terpenes identified and showing high variation
under UV-B were the sesquiterpene E-nerolidol (7) and the monoterpenes
a-pinene (8), terpinolene (9) and carene (10). These
metabolites are produced by plants at low concentrations and their
production was increased in the presence of UV-B, more associated
with high UV-B and in young leaves. However, in the case of the
monoterpenes, they were not detected in control plants suggesting
that they were synthesized de novo as a result of UV-B elicitation.
In this sense, in previous work, it was found that this radiation regulated
expression of genes encoding TPS (Pontin et al., 2010). These
substances are highly lipophylic and possess good antioxidative
capacity in the lipophilic test systems. Graßmann et al. (2001,
2003) demonstrated that terpinolene (9) protects low-density lipoprotein
from oxidation. It is well-known that in vegetative tissues ABA (11) levels increase
when plants are exposed to adverse environmental conditions,
such as drought, salt and temperature stress (Crozier et al.,
2000). In the present experiments, and in agreement with previous
results (Berli et al., 2010) ABA (11) levels increased in response to
UV-B following a dose–response behavior according to the fluence
rate of UV-B and in the young leaves. However, no increase in
expression of genes involved in ABA (11) synthesis was previously
found (Pontin et al., 2010). Thus, increased ABA (11) levels could be
explained by hydrolysis of glycosylated ABA from vacuoles in
plants exposed to UV-B, since ABA (11) levels in UV-B treated tissues
experiment a sharp decrease 24 h post irradiation, even respect
to non-irradiated controls. In young leaves, ABA (11) levels
decreased 21.2-fold when submitted to high UV-B and 9.4-fold in
low UV-B irradiation, regarding the values obtained immediately
after the irradiation treatments had finished. Furthermore, in
mature leaves they decreased 5.7-fold in high UV-B and 8.3-fold
at low UV-B irradiation. Such a decay in ABA (11) levels below
the controls could be explained by an increase in its turnover,
which correlate fairly well with the induction of a gene encoding
ABA 80-hydroxylase in UV-B treated plants (the major ABA catabolic
pathway in higher plants; Pontin et al., 2010).
Several experiments have shown that volatile isoprenoids protect
leaves against abiotic stresses as burst of heat (Delfine et al.,
2000; Loreto et al., 1998; Sharkey and Singsaas, 1995; Singsaas
et al., 1997) and ozone (Loreto et al., 2001), suggesting that isoprenoids
are effective antioxidants in leaves. Also, they are proposed
to act as chloroplast membranes stabilizers, a role that has been
assigned to other isoprenoids, for example xanthophylls and
a-tocopherol (5) (Havaux, 1998; Sharkey, 1996). Many volatile
plant monoterpenes and sesquiterpenes combine rapidly with
ROS (Calogirou et al., 1999), and their emission is stimulated by
high light and temperature conditions (Delfine et al., 2000; Duhl
et al., 2008). Thus, these compounds might also be involved in
resistance to abiotic stress. In this study, treated plants showed
that E-nerolidol (7), pinene (8), terpinolene (9) and carene (10) increased
either under high UV-B as well as low UV-B irradiation
when compared to control plants. These results are comparable
to those obtained by Chapman (2009) in lemon verbena (Aloysia
citriodora), where higher leaf concentrations of monoterpenes were
found when grown without protection to UV-B exposure as comcompared
to plants with UV-B exclusion. In the present work, maximum
accumulation of both mono and sesquiterpenes was
observed in plants treated with high UV-B. Thus, induction of these
compounds could be interpreted as a mechanism in order to protect
grapevine plants against UV-B-induced oxidative damage.
Our results suggest that endogenous terpenes may have an important
antioxidant role in plants since their levels are increased as
the tissues are exposed to high UV-B irradiation.