The Role of Homogentisate Phytyltransferase and Other,
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The Role of Homogentisate Phytyltransferase and Other
Tocopherol Pathway Enzymes in the Regulation of
Tocopherol Synthesis during Abiotic Stress
Eva Collakova and Dean DellaPenna*
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,
Michigan 48824
Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change
significantly during plant growth and development and in response to stress, likely as a consequence of the altered
expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosyn-
thesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632–642). Wild-type and
transgenic Arabidopsis plants constitutively overexpressing HPT (35S::
HPT1
) were subjected to a combination of abiotic
stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were
determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::
HPT1
leaves, respectively, with tocopherol levels in 35S::
HPT1
being 2- to 4-fold higher than wild type at all experimental time
points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::
HPT1
and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition,
substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis
during stress. The accumulation of high levels of
-,
-, and
-tocopherols in stressed tissues suggested that the methylation
of phytylquinol and tocopherol intermediates limit
-tocopherol synthesis. Overexpression of
-tocopherol methyltrans-
ferase in the 35S::
HPT1
background resulted in nearly complete conversion of
- and
-tocopherols to
- and
-tocopherols,
respectively, indicating that
-tocopherol methyltransferase activity limits
-tocopherol synthesis in stressed leaves.
Tocopherols are a group of lipid soluble antioxi-
dants collectively known as vitamin E that are essen-
tial components of animal diets. Dietary vitamin E is
required for maintaining proper muscular, immune,
and neural function and may be involved in reducing
the risk of cancer, cardiovascular disease, and cata-
racts in humans (Pryor, 2000; Brigelius-Flohe et al.,
2002). In plants, tocopherols are believed to protect
chloroplast membranes from photooxidation and
help to provide an optimal environment for the pho-
tosynthetic machinery (Fryer, 1992; Munne-Bosch
and Alegre, 2002a). Many of the proposed tocopherol
functions in animals and plants are related to their
antioxidant properties, the most prominent of which
is protection of polyunsaturated fatty acids from
lipid peroxidation by quenching and scavenging var-
ious reactive oxygen species (ROS) including singlet
oxygen, superoxide radicals, and alkyl peroxy radi-
cals (Fukuzawa and Gebicky, 1983; Munne-Bosch
and Alegre, 2002a).
Tocopherols are only synthesized by photosyn-
thetic organisms and consist of a polar chromanol
ring and a 15-carbon lipophilic prenyl chain derived
from homogentisic acid (HGA) and phytyl diphos-
phate (PDP; Fig. 1). In plants, HGA is formed from
p
-hydroxyphenyl pyruvate (HPP) by the cytosolic
enzyme HPP dioxygenase (HPPD; Garcia et al., 1997,
1999; Norris et al., 1998). On the basis of radiotracer
studies, HPP can originate either from prephenate or
Tyr by the shikimate pathway, but the relative con-
tribution of these two precursors to the total HPP
pool is unknown (Threlfall and Whistance, 1971;
Fiedler et al., 1982; Lopukhina et al., 2001). In plas-
tids, isopentenyl diphosphate derived from the 1-de-
oxyxylulose-5-phosphate (DXP) pathway (Eisenreich
et al., 1998; Lichtenthaler, 1998) is used by gera-
nylgeranyl diphosphate synthase 1 (GGPS1) for the
synthesis of geranylgeranyl diphosphate (GGDP;
Okada et al., 2000). Three of the four double bonds in
the GGDP molecule are reduced to form PDP
through partially reduced intermediates by a multi-
functional GGDP reductase (GGDR; Addlesee et al.,
1996; Keller et al., 1998). Alternatively, PDP can be
generated from phytol and ATP by a kinase activity
present in chloroplast stroma (Soll et al., 1980).
Homogentisate phytyltransferase (HPT) is a
membrane-bound chloroplast enzyme, which cata-
lyzes the committed step of tocopherol biosynthesis,
the condensation of HGA and PDP, to form 2-
methyl-6-phytyl-1,4-benzoquinol (MPBQ; Soll,1987;
Collakova and DellaPenna, 2001; Savidge et al.,
2002). MPBQ can be methylated to 2,3-dimethyl-6-
phytyl-1,4-benzoquinol (DMPBQ) by MPBQ methyl-
transferase (MPBQ MT; Marshall et al., 1985; Soll,
1987; Shintani et al., 2002). MPBQ and DMPBQ can be
* Corresponding author; e-mail dellapen@msu.edu; fax 517–
353–9334.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026138.
930
Plant Physiology
, October 2003, Vol. 133, pp. 930–940, www.plantphysiol.org © 2003 American Society of Plant Biologists
Regulation of Tocopherol Synthesis in Arabidopsis
-tocopherol levels are observed during aging
and senescing of plants (Rise et al., 1989; Molina-
Torres and Martinez, 1991; Tramontano et al., 1992),
possibly to protect cellular components from in-
creased oxidative stress (Munne-Bosch and Alegre,
2002b). Enhanced tocopherol accumulation also oc-
curs in response to a variety of abiotic stresses in-
cluding high light, drought, salt, and cold and may
provide an additional line of protection from oxida-
tive damage (Havaux et al., 2000; Munne-Bosch and
Alegre, 2002a).
Although there is a growing body of knowledge
about the individual enzymes required for tocoph-
erol biosynthesis in plants, the mechanisms that reg-
ulate the overall pathway and result in differential
tocopherol content and composition during plant de-
velopment or stress remain poorly understood. Reg-
ulation of tocopherol biosynthesis in senescing and
stressed plants may occur at multiple steps of the
pathway. HPPD activity limits tocopherol synthesis
in non-stressed Arabidopsis plants (Tsegaye et al.,
2002), and HPPD mRNA levels are up-regulated in
senescing barley (
Hordeum vulgare
) leaves (Klebler-
Janke and Krupinska, 1997). Similarly, various biotic
and abiotic stresses elevate Tyr aminotransferase
(TAT) mRNA and protein levels and enzyme activity
in Arabidopsis (Lopukhina et al., 2001; Sandorf and
Hollander-Czytko, 2002). Whether other steps of the
tocopherol pathway are also involved in the regula-
tion of tocopherol biosynthesis during stress remains
to be determined.
It has been recently demonstrated that HPT activity
limits tocopherol synthesis in non-stressed Arabi-
dopsis leaves (Collakova and DellaPenna, 2003). The
gene encoding HPT,
HPT1
, has been cloned from
Synechocystis
sp. PCC 6803 and Arabidopsis (Colla-
kova and DellaPenna, 2001; Schledz et al., 2001).
Overexpression of HPT in Arabidopsis increased leaf
and seed tocopherol content by up to 4.4-fold and
75%, respectively (Savidge et al., 2002; Collakova and
DellaPenna, 2003). The current study was undertaken
to further define the role of HPT in regulating tocoph-
erol biosynthesis in stressed photosynthetic tissues. By
combining abiotic stress with molecular and bio-
chemical analyses, we have also identified additional
enzymes and/or substrates that limit
Figure 1.
Tocopherol biosynthesis in plants. Dashed arrows repre-
sent multiple steps. Enzymes are indicated in circles: HPT; TAT; PD,
prephenate dehydrogenase; HPPD; HGAD; GGDR; GGPS1; KIN,
unspecified kinase; CHLase, chlorophyllase; MPBQ MT; TC;
-TMT.
cyclized by tocopherol cyclase (TC) to form
- and
-tocopherol, respectively (Stocker et al., 1996;
Arango and Heise, 1998; Porfirova et al., 2002). The
last enzyme of the pathway,
-tocopherol
-tocopherol methyl-
synthesis in stressed Arabidopsis leaves.
transferase (
-TMT), catalyzes methylation of
- and
-tocopherol, respectively
(D’ Harlingue and Camara, 1985; Shintani and
DellaPenna, 1998).
In plants, tocopherol levels and composition vary
in different tissues and fluctuate during development
and in response to abiotic stresses. Dry and germi-
nating seeds of many plants accumulate predomi-
nantly
- and
RESULTS
Biochemical and Physiological Responses of
Wild-Type and 35S::
HPT1
Plants to Abiotic Stress
-tocopherol is the ma-
jor tocopherol in leaves, which may reflect distinct
Stress is associated with increased total tocopherol
levels in a variety of plants (for review, see Munne-
Bosch and Alegre, 2002a). We have shown previously
that HPT activity is limiting for tocopherol synthesis
Plant Physiol. Vol. 133, 2003
931
roles of individual tocopherols in these tissues
(Bramley et al., 2000; Franzen and Haas, 1991;
Shintani and DellaPenna, 1998). Significant increases
in leaf
-tocopherol to
-tocopherol, whereas
Collakova and DellaPenna
in non-stressed Arabidopsis leaves (Collakova and
DellaPenna, 2003). To investigate whether HPT activ-
ity also limits tocopherol synthesis in stressed Ara-
bidopsis leaf tissue, 6-week-old wild type and two
well-characterized 35S::
HPT1
lines (lines 11 and 54;
Collakova and DellaPenna, 2003) were subjected to a
combination of nutrient deficiency and high-light
stress (0.8–1 mmol photons m
2
s
1
) for up to 15 d,
and tocopherol content and composition were ana-
lyzed during the treatment.
Total tocopherol levels increased in a near linear
manner during exposure of both wild-type and
35S::
HPT1
plants to stress (
R
2
0.05
nmol cm
2
leaf area, respectively). In response to 15 d
of abiotic stress, total tocopherol levels increased to
16.4
0.3 nmol cm
2
leaf area in wild-type plants. In con-
trast, 15 d of growth in the absence of stress increased
tocopherol levels in wild-type and 35S::
HPT1
leaves
less than 2-fold to 0.49
0.50 nmol
cm
2
leaf area, respectively, most likely as a result of
aging. Aging has previously been associated with a
moderate increase in leaf tocopherol content in a va-
riety of plants (Rise et al., 1989; Molina-Torres and
Martinez, 1991; Tramontano et al., 1992). In 8-week-
old plants, the overall increase in total tocopherol
levels in stressed relative to non-stressed plants was
18- and 8-fold for wild type and 35S::
HPT1
, respec-
tively. At any time point during stress treatments,
total tocopherol levels in 35S::
HPT1
were 1.9- to 3.8-
fold higher than wild type (
P
0.97; Fig. 2A). Before
0.006, Fig. 2A), sug-
gesting that HPT activity limits tocopherol synthesis
in stressed wild-type Arabidopsis leaves.
The general response to stress was monitored by
assessing anthocyanin accumulation and alterations
to chlorophyll and carotenoid levels (Fig. 2, B and C).
Total anthocyanin levels rapidly increased from be-
low detection to approximately 35
mol cm
2
leaf
area by d 6 and were maintained at this level
throughout the stress treatment (Fig. 2B). Total chlo-
rophyll and carotenoid levels decreased gradually
during the 15-d stress treatment to approximately
half of their initial levels (Fig. 2C). There were no
significant differences in chlorophyll, carotenoid, or
anthocyanin content between wild type and
35S::
HPT1
throughout the course of the experiment
(Fig. 2, B and C). It appears that the excess tocopherol
accumulated in 35S::
HPT1
leaves does not afford ad-
ditional protection of chlorophylls and carotenoids
during stress. Detailed analyses of membrane lipids
and their oxidation products under stress conditions
are required to more directly address the issue of
tocopherol functions in plants.
Other than elevated tocopherol levels, there were
no obvious phenotypic differences between wild-
type and transgenic plants under normal or stressed
conditions. Regardless of genotype, growth of all
plants subjected to abiotic stress was inhibited rela-
tive to control plants (data not shown), most likely
due to a combination of the over-reduced photosys-
tems, oxidative stress, and nutrient deficiency. Be-
cause all plants were grown at a 10-h photoperiod,
bolting and flowering did not occur during the ex-
perimental time frame.
Figure 2.
Total tocopherol, anthocyanin, chlorophyll, and carot-
enoid levels in stressed wild-type and 35S::
HPT1
leaves. Plants of the
indicated genotypes were grown in a 10-h/14-h light/dark cycle at 75
to 100
mol photons m
2
s
1
for 6 weeks and then transferred to
mol photons m
2
s
1
growth conditions. A,
Total tocopherol levels in leaves of stressed wild-type and 35S::
HPT1
plants. High-light stress resulted in a significant elevation of total
tocopherol levels in both 35S::
HPT1
transgenic and wild-type plants.
B, Anthocyanin accumulation in leaves of stressed wild-type and
35S::
HPT1
Arabidopsis plants. Anthocyanin levels increased within
the first3dofstress and reached high steady-state levels after6dof
stress treatment. C, Chlorophyll and carotenoid degradation in leaves
of stressed wild-type and 35S::
HPT1
plants. Total chlorophyll and
carotenoid levels decreased gradually to approximately 50% of the
initial levels in all stressed lines.
HPT Expression and Enzyme Activity in
Unstressed and Stressed Wild Type and 35S::HPT1
We have shown previously that non-stressed
35S::
HPT1
Arabidopsis plants accumulated 20- to
932
Plant Physiol. Vol. 133, 2003
stress treatment, the total tocopherol levels of 6-week-
old 35S::
HPT1
plants were 3-fold higher than the cor-
responding wild type (1.06
0.22 and 0.36
0.8 nmol cm
2
leaf area in 35S::
HPT1
and 8.7
0.04 and 2.00
approximately 900
Regulation of Tocopherol Synthesis in Arabidopsis
100-fold higher
HPT1
mRNA levels than wild type
and showed 4- to 10-fold increases in HPT specific
activity, which resulted in up to 4.4-fold increased
tocopherol levels in leaves of transgenic lines com-
pared with wild type (Collakova and DellaPenna,
2003). In non-stressed wild-type leaves, average HPT
mRNA levels ranged from 0.3 to 0.6 fmol mg
1
total
RNA during the 12-d experimental time course (Fig.
3A). Consistent with our previous study (Collakova
and DellaPenna, 2003), HPT mRNA levels in non-
stressed 35S::
HPT1
leaves were at least 20-fold higher
Figure 4.
Relative HPT specific activity in control and stressed wild-
type and 35S::
HPT1
Arabidopsis chloroplasts. Six-week-old plants
were transferred to high light (0.8–1 mmol photons m
2
s
1
) for 3
and 6 d, and chloroplasts were isolated and assayed for HPT activity.
Results from two independent experiments are presented as an av-
erage
0.10 pmol h
1
mg
1
protein). Abiotic stress
resulted in the strong induction of HPT activity relative to control
plants in both wild type and 35S::
HPT1
. Relative HPT specific ac-
tivity in 35S::
HPT1
was significantly higher (
P
0.0005) than in wild
type at all time points.
than wild type and ranged from 7 to 15 fmol mg
1
total RNA during the course of the experiment
(Fig. 3B).
To assess any correlation between elevated total
tocopherol levels and changes in HPT expression or
activity during abiotic stress, HPT mRNA levels and
specific activity were determined in non-stressed and
stressed wild-type and 35S::
HPT1
plants. HPT
mRNA levels were significantly elevated up to 3.5-
fold in wild type after3dofhigh-light treatment and
remained elevated throughout the course of the ex-
periment (Fig. 3A;
P
0.0005). In response to 3
and6dofstress, HPT specific activity in wild-type
and 35S::
HPT1
lines increased approximately 3-fold
and up to 4.4-fold relative to their respective un-
stressed controls. In 35S::
HPT1
, the relative HPT spe-
cific activity after3dofstress was 9-fold that of
comparably treated wild type. After6dofstress,
HPT specific activity was 5.6- and 3.5-fold higher
than the corresponding wild type for 35S::
HPT1
lines
11 and 54, respectively (Fig. 4).
Figure 3.
HPT expression in control and stressed wild-type and
35S::
HPT1
plants. Plants were grown and stressed as described in
Figure 2, total RNA was extracted, and HPT mRNA levels determined
by real-time PCR. Data are normalized for EF-1
mRNA levels and
SD
of three independent experiments. A,
Wild-type HPT mRNA levels. B, Wild-type and 35S::
HPT1
HPT
mRNA levels. Stress resulted in an up-regulation of HPT mRNA levels
in wild type, whereas no trend was observed in stressed 35S::
HPT1
and the corresponding control plants. * and ** represent
P
0.05
and 0.01, respectively.
Plant Physiol. Vol. 133, 2003
933
SD
of the activity increase relative to wild-type non-stressed
chloroplasts (0.15
0.05). No clear trend was
observed for HPT mRNA levels in stressed
35S::
HPT1
lines (Fig. 3B), although there was signif-
icant biological variation in HPT mRNA levels in
both wild-type and 35S::
HPT1
plants during stress
treatments (Fig. 3, A and B). Consistent with prior
studies, HPT specific activity in the absence of abiotic
stress was 6-fold higher in 35S::
HPT1
lines compared
with wild type (Fig. 4;
P
presented as average
Collakova and DellaPenna
Changes in mRNA Levels of Other Tocopherol
Pathway-Related Genes in Non-Stressed and Stressed
Wild-Type and 35S::HPT1 Leaves
leaves using real-time PCR (Fig. 5). TAT and HPPD
catalyze formation of the tocopherol biosynthetic
precursors HPP and HGA, respectively, whereas
HGAD is involved in HGA degradation (Fig. 1).
GGPS1 and GGDR catalyze synthesis of PDP, a pre-
nyl substrate used in tocopherol biosynthesis,
whereas TC and
-TMT) may play roles in regulating
tocopherol synthesis in Arabidopsis leaves. Consis-
tent with our previous study (Collakova and Della-
Penna, 2003), there were no significant differences
between genotypes for mRNA levels of these genes
in stressed or unstressed plants (Fig. 5). These results
indicate that increased HPT expression in 35S::
HPT1
transgenic lines does not significantly impact the ex-
pression of other tocopherol pathway-related genes.
To assess the role of these enzymes in regulating
tocopherol accumulation during stress, their mRNA
levels were measured in wild-type and 35S::
HPT1
-TMT are involved in regulating
tocopherol composition (Fig. 1). The steady-state
mRNA levels of TAT, HPPD, HGAD, GGPS1, and
-TMT in non-stressed tissues were similar in the
different genotypes and varied between 1 and 6 fmol
mg
1
total RNA during the 12-d experimental time
course (Fig. 5, A, B, C, D, and G). GGDR showed the
highest steady-state mRNA levels of all tested genes
and fluctuated between 20 and 30 fmol mg
1
total
RNA in non-stressed Arabidopsis leaves (Fig. 5E). TC
mRNA levels were quite low (0.3–0.6 fmol mg
1
total
Figure 5.
Expression of other tocopherol-related
genes in control and stressed wild-type and
35S::
HPT1
leaves. Experiments were performed
as described in Figure 3. A, TAT; B, HPPD; C,
HGAD; D, GGDR; E, GGPS1; F, TC; G,
-TMT
mRNA levels were not up-regulated in response
to stress. * indicates all three stressed genotypes
were statistically different (
P
0.05) from their
corresponding unstressed controls at the indi-
cated time points except for HGAD at d 3,
where only wild type and 35S::
HPT1
-11 mRNA
levels were statistically different from the corre-
sponding controls.
934
Plant Physiol. Vol. 133, 2003
In addition to HPT, several other tocopherol bio-
synthetic enzymes (TAT, HPPD, HGAD, GGPS1,
GGDR, TC, and
-TMT.
TAT, HPPD, and HGAD mRNA levels in-
creased, whereas GGDR mRNA levels de-
creased during stress. GGPS1, TC, and
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