Download An Arabidopsis Mutant Tolerant to Lethal

An Arabidopsis Mutant Tolerant to Lethal Ultraviolet-B
Levels Shows Constitutively Elevated Accumulation of
Flavonoids and Other Phenolics1
Kim Bieza2 and Rodrigo Lois*
Department of Biological Science, California State University, 800 North State College Boulevard, Fullerton,
California 92834
The isolation and characterization of mutants hypersensitive to ultraviolet (UV) radiation has been a powerful tool to learn
about the mechanisms that protect plants against UV-induced damage. To increase our understanding of the various
mechanisms of defense against UVB radiation, we searched for mutations that would increase the level of tolerance of
Arabidopsis plants to UV radiation. We describe a single gene dominant mutation (uvt1) that leads to a remarkable tolerance
to UVB radiation conditions that would kill wild-type plants. Pigment analyses show a constitutive increase in accumulation
of UV-absorbing compounds in uvt1 that increases the capacity of the leaves to block UVB radiation and therefore is likely
to be responsible for the elevated resistance of this mutant to UVB radiation. These increases in absorption in the UV region
are due, at least in part, to increases in flavonoid and sinapate accumulation. Expression of chalcone synthase (CHS) mRNA
was shown to be constitutively elevated in uvt1 plants, suggesting that the increases in absorption may be a consequence of
changes in gene expression. Expression of CHS in uvt1 was shown to be still inducible by UV, indicating that the uvt1 lesion
may not affect the UV-mediated regulation of CHS gene expression. Our data support an important role for UV screens in
the overall protection of plants to UVB radiation. The uvt1 mutant could prove to be an important tool to elucidate further
the exact role of UV-absorbing pigments in UV protection as well as the relative contribution of other mechanisms to the
overall tolerance of plants to UV radiation.
Plants, in their need to capture sunlight for photosynthesis, are unavoidably exposed to the damaging
effects of UVB radiation. The highly energetic photons in these wavelengths cause damage to DNA and
other macromolecules, which can lead to cellular injury, mutagenesis, and death. DNA is one of the
major targets of UV damage; however, other components (e.g. proteins, membrane lipids, etc.) also appear to be damaged by UV. For example, UV radiation is known to affect protein synthesis (Andley et
al., 1990; Hightower et al., 1994), to directly damage
proteins through absorption in the UVB region by
aromatic aminoacids (Jagger, 1985; Pigault and Gerard, 1989; Dillon, 1991; Caldwell, 1993), to have effects on enzymatic function (Jordan et al., 1992; Pfundel and Pan, 1992), and to damage lipids through
peroxidation (Taira et al., 1992; Hightower et al.,
1994). Part of the damage induced by UVB is believed
1
This work was supported by the National Institute of Environmental Health Sciences, National Institutes of Health (grant no.
R29 –ES07575). Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
National Institute of Environmental Health Sciences, National Institutes of Health. This work was also supported by the California
State University Special Fund for Research Scholarship and Creative Activity.
2
Present Address: The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, CA 92037.
* Corresponding author; e-mail [email protected]; fax
714 –278 –5044.
to be caused indirectly through the production of free
radicals, such as superoxide radicals, singlet oxygen,
and hydroxyl radicals (Pathak and Stratton, 1969;
Peak and Peak, 1975; Peak and Peak, 1983; Foyer et
al., 1994). A variety of adaptations have evolved that
help plants cope with the exposure to UV but we do
not yet understand the exact role many of them play
in the overall protection against UV damage. The
isolation and characterization of mutants has been a
powerful tool to learn about various mechanisms
that help protect plants against different types of UV
radiation damage. Mutants showing hypersensitivity
to UV radiation have been instrumental in laying
down the groundwork for our understanding of
some of these mechanisms. For example, studies of
the transparent testa, fah1, and uvs mutants (Li et al.,
1993; Lois and Buchanan, 1994; Landry et al., 1995)
have increased our understanding of different aspects of the role of phenolics, including flavonoids
and sinapates, in the protection of plants against UV
radiation (for review, see Bharti and Khurana, 1997).
Mutants deficient in DNA repair (Britt et al., 1993;
Jiang et al., 1997; Vonarx et al., 1998) have helped
elucidate the involvement of mechanisms of photoreactivation and dark repair of DNA in UV
protection.
In an attempt to increase our understanding of the
various mechanisms of defense against UVB radiation, we set out to search for mutations that would
increase, rather than decrease, the level of tolerance
of Arabidopsis plants to UV radiation. We describe
Plant Physiology, July 2001, Vol. 126,
pp. 1105–1115,
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Bieza and Lois
below the isolation and preliminary characterization
of an Arabidopsis mutant that shows a remarkable
tolerance of UV radiation levels that are lethal to
wild-type plants. Our data indicates that a constitutive increase in accumulation of UV-absorbing pigments due to changes in gene expression is responsible for this elevated resistance to UVB. This mutant
may prove to be a powerful tool to further elucidate
the molecular mechanisms of protection against UV
as well as the potential contribution of UV screens
and other mechanisms to the overall tolerance of
plants to UV radiation.
RESULTS AND DISCUSSION
Isolation of the uvt1 Mutant
With the goal of increasing our understanding of
the different mechanisms of protection against UVB
radiation in Arabidopsis, we set out to isolate mutants that show elevated tolerance to UV. To increase
our chances of detecting increases in UV tolerance,
we used a mutant highly sensitive to UV radiation
(uvs), previously isolated in our laboratory, as the
genetic background for a new round of ethyl methane sulfonate (EMS)-mediated mutagenesis. As described previously (Lois and Buchanan, 1994), the UV
sensitivity of uvs is due to a single recessive lesion in
a gene that leads to altered accumulation of UVabsorbing compounds. This mutant does not show
the significant increase in absorption in the UV range
observed in the wild type upon exposure to UVB
radiation. We believe that the increased levels of
these UV-absorbing compounds act as a shield that
blocks UV before it reaches sensitive targets and that
the lack of these derivatives in uvs results in this
mutant’s extreme sensitivity to UV light.
After EMS mutagenesis, M1 generation uvs seedlings were screened during UV irradiation for plants
that showed diminished UV damage as evidenced by
greener, healthier appearance than the rest of the uvs
population. uvs plants typically become necrotic after
a short exposure to low levels of UVB. A screening of
approximately 5,000 EMS-treated seeds led to the
isolation of three mutants exhibiting a pronounced
increase in tolerance to UV, which we have named
uvt1, uvt2, and uvt3 (for UV tolerant). The mutant
uvt1 showed the most dramatic increase in tolerance
to UVB and is described below.
Because uvt1 was isolated in a screening of the M1
population, it was unlikely that we would isolate
any recessive mutations. Thus, from a selfing cross
of the uvt1 mutant we expected to find a segregating
population of resistant and sensitive plants. Furthermore, because the M1 population consists of
chimeric plants harboring the mutation only in certain clonal sectors, it was clear that the uvt1 mutation would be passed on to the next generation only
if the mutated sector included the gamete-forming
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cells. The progeny from a uvt1 self-cross segregated
into two distinct populations of plants of UVsensitive and UV-resistant phenotypes in a ratio
consistent with that of a dominant heritable characteristic caused by a lesion in a single Mendelian
locus (data not shown). Figure 1A shows the effects
of irradiating uvt1 and uvs seedlings with UVB.
Under these conditions, we observed no signs of
damage in uvt1 plants, whereas the typical severe
necrosis was clearly evident in uvs. We subsequently crossed the uvt1 mutant to wild-type plants
and allowed the resulting progeny to self. The F2
population segregated with respect to UV resistance
(Fig. 1B). We observed individuals displaying three
distinct phenotypes, a uvt1-like phenotype (top), a
uvs-like phenotype (bottom), and an intermediate
UV tolerance phenotype (center) that later proved to
have wild type-like UV tolerance. Because the original uvt1 mutant also carried a gl1 genotype, after
the backcross the population is segregating with
respect to both uvt1 and gl1 phenotypes. Thus, it is
important to note that the UV-tolerant phenotype is
observed in plants with or without trichomes (Fig.
1C). The same is true of the UV-sensitive and wildtype phenotypes (not shown). This suggests that the
intrinsic differences in UV tolerance between these
plants are not significantly affected by the presence
or absence of trichomes in their leaves.
From these populations segregating for uvt1, gl1,
and uvs, we obtained uvt1 plants in a wild-type genetic background for both the gl1 and uvs loci. The
level of UV tolerance of the uvt1/uvs double mutant,
however, appears not to be significantly different
from that of uvt1 plants in a wild-type background
(data not shown). Figure 2 shows an assessment of
the UV tolerance of mature plants from uvs, wild-type
controls, and uvt1 mutants (wild-type background).
As previously described (Lois and Buchanan, 1994),
under identical UVB irradiation conditions, the uvs
mutant showed more pronounced necrotic lesions
than the wild type. The uvt1 mutant, however,
showed significantly higher UVB tolerance than both
uvs and the wild type. This is especially true under
high intensity UVB irradiation conditions (high UV)
that are well tolerated by uvt1 plants but that are lethal
to both uvs and the wild type. From its extreme UVB
tolerance, we can infer that uvt1 cannot be simply a
revertant from uvs to wild-type genotype, but rather a
new mutation that leads to hyper-resistance to UV.
Furthermore, it appears that the elevated tolerance of
uvt1 plants is evident at both early (Fig. 1) and late
stages of development (Fig. 2) and in both young and
older leaves. In UV-irradiated wild-type Arabidopsis,
one normally observes a gradient of damage whereby
older leaves are more damaged than younger leaves
(Lois, 1994). This appears not to be the case in uvt1
where all leaves, including the oldest leaves, are
highly tolerant to UV radiation.
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Plant Physiol. Vol. 126, 2001
Arabidopsis Mutant Hyper-Tolerant to UVB Radiation
Figure 1. Effect of UVB-radiation on UV-sensitive and UV-tolerant seedlings of Arabidopsis (A) and on a segregating
population of uvt1/uvs seedlings (B and C). A, Twelve-day old uvs and uvt1 plants were irradiated with 0.17 W m⫺2 UVB
for 48 h and allowed to recover for 48 h under fluorescent lighting before being photographed. Representative individuals
of UV-tolerant (uvt1) and UV-sensitive (uvs) plants are shown. B, Seventeen-day old seedlings from the F2 generation of a
cross between wild-type and uvt1 plants were exposed to 0.17 W m⫺2 UVB for 37 h and allowed to recover for 27 h under
fluorescent lighting before being photographed. Representative individuals of each of three distinct UV tolerance phenotypes
observed, highly tolerant (uvt1), intermediate tolerance (WT) ,and low tolerance (uvs), are shown. C, Additional seedlings
of the same experiment are shown to depict the segregation of the glabrous mutation (gl1) among uvt1 plants. All images
were digitized before printing.
Increased UV Tolerance and Dark-Green Phenotypes in
uvt1 Are Likely Due to Constitutively Elevated
Flavonoid Accumulation
The uvt1 mutation leads to different phenotypes
that may or may not be related to the effects it has on
UV tolerance. These include significantly shortened
hypocotyls, a slower rate of growth, and delayed
flowering with respect to wild-type plants. Some of
these changes suggest that the defect in uvt1 may
involve components of the light signal transduction.
On the other hand, uvt1 seedlings show a phototropic
response indistinguishable from the wild type (data
not shown). As can be observed in Figure 2, leaves
from uvt1 plants also appear to be significantly
darker than those in wild-type or uvs plants, especially in the petioles and midribs of UV-exposed
plants, which show a purple coloration. These observations indicated that the increased UV tolerance of
uvt1 might be related to increased pigmentation.
Therefore, we investigated the possibility that the
darker coloration and UV-tolerant phenotypes may
be due to the same genetic lesion and would therefore segregate together. After scoring and marking
586 dark plants, according to their light and dark
Plant Physiol. Vol. 126, 2001
coloration, these plants were exposed to UV radiation and analyzed for their sensitivity to UV. As
expected, almost all plants showed tolerance to UV.
Only three among the 586 plants marked as dark
were found to show a UV-sensitive phenotype. Because we cannot rule out that these three plants may
have been erroneously marked as dark, from this
data we conclude that the two phenotypes are very
tightly linked and may even be caused by the same
genetic lesion.
A purple coloration in leaf tissues is commonly the
result of an accumulation of the pigmented flavonoids, anthocyanins (Holton and Cornish, 1995). It
seemed likely then that an increased level of flavonoids in the uvt1 mutant might be responsible for
its dark coloration. By the same token, since flavonoids and other UV-absorbing phenolics have
been postulated as important UV defense mechanisms (Li et al., 1993; Lois, 1994; Lois and Buchanan,
1994; Landry et al., 1995; Mazza et al., 2000), it appeared possible that the increased UV resistance of
uvt1 may be due to the increase in UV-absorbing
compounds. To address these questions, we investigated if the levels of these pigments were higher in
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Bieza and Lois
Figure 2. Effect of different regimes of UVB irradiation on mature wild-type and mutant Arabidopsis plants. One-month-old uvt1, wild-type
(WT), and uvs plants were exposed to 0.10 W
m⫺2 UVB (mid UV), 0.18 W m⫺2 UVB (high
UV), or kept under fluorescent lighting (no UV)
for 3 d and allowed to recover for 3 d under
fluorescent lighting before being photographed.
This image was digitized before printing.
the uvt1 mutant than in uvs and wild-type controls.
Normally, wild-type plants accumulate low levels of
UV-absorbing pigments, and these levels increase
significantly upon UVB exposure. The absorption
spectra in Figure 3 are an extension of data presented
previously by Lois and Buchanan (1994) and show
the normal UV-mediated increase in UV absorption
in the region between 260 and 350 nm in wild-type
controls, as well as the minimal increase in UV absorption in this region in uvs when plants are exposed to UVB. In contrast, the absorption spectrum
of uvt1 shows elevated levels of UV-absorbing pigments prior to UV irradiation. A similar result was
observed when data for extract absorption at 330 nm
(the peak of absorption in Fig. 3) from three independent experiments were pooled (Fig. 4). Again, we
observe the normal UV-mediated increase in absorption at 330 nm in the wild type, and the minimal
increase in uvs. We also see significantly elevated
levels of UV-absorbing compounds in nonexposed
uvt1 when compared with uvs and wild-type controls. We conclude from these data that before UV
exposure the levels of UV-absorbing pigments in uvt1
tissue are significantly higher than those in uvs and
wild type, and that they are comparable to the levels
seen in wild type after UV exposure. This in turn
indicates that the cause of the increased UV tolerance
of uvt1 might be mediated by the increased accumulation of UV-absorbing pigments. This is supported
by absorption studies in the progeny of a selfing
cross of a uvt1 heterozygote. When analyzing extracts
from over 50 individual UV-tolerant and UVsensitive plants from the F1 generation we obtained
in all cases, low absorption value at 330 nm (A330) for
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individual UV-sensitive plants and high levels for
uvt1 plants (data not shown). Therefore, the simplest
explanation for the data presented is that the high
level of pigment absorption at 330 nm is the cause of
the elevated resistance of uvt1 to UV radiation. In this
light, the fact that uvt1 plants show constitutive accumulation of UV-absorbing pigments before exposure to UV might explain why this mutant is even
more UV resistant than the wild type. The high level
of UV-absorbing pigments in uvt1 at the time of
initial exposure to UV may protect uvt1 plants from
early UV-mediated damage. On wild-type Arabidopsis plants, detectable increases in flavonoid accumulation begin only after about 7 h of UV exposure
(Lois, 1994). Thus, damaging levels of UV radiation
may be able to penetrate (and damage) the leaf for
some time until the accumulation of UV-screening
compounds is above a threshold of effective
protection.
In the absorption spectra in Figure 3, one can also
observe higher levels of absorption for the peaks in
the 400- to 500-nm and the 650-nm regions. Because
these peaks correspond to the absorption by carotenoids and chlorophyll, it would appear that uvt1
might accumulate higher levels of these pigments in
addition to the pigments absorbing in the 330-nm
range. Pooling the data from three independent experiments using a larger number of plants, however,
we have been unable to show that the elevated levels
of carotenoids and chlorophyll between uvt1 and
wild-type plants are statistically significant (data not
shown).
We have hypothesized above that the dark-green
phenotype characteristic of uvt1 (Fig. 2) may be due
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Plant Physiol. Vol. 126, 2001
Arabidopsis Mutant Hyper-Tolerant to UVB Radiation
Figure 4. Level of UV-absorbing pigments in mutant and wild-type
Arabidopsis plants. Pigments were extracted in 80% (v/v) ethanol
from aerial tissues of 13-d-old plants before (⫺) and after (⫹) 21 h
exposure to 0.15 W m⫺2 UVB -radiation. The height of the bars
represents the mean value of A330 per milligrams tissue of three
independent experiments. Error bars correspond to one SD. Lines
below bars depict relevant cases of pairs of bars for which there is a
statistically significant difference between their means (non-paired
Student’s t test, P ⬍ 0.05).
Figure 3. Changes in the absorption spectra of pigment extracts from
uvs, uvt1, and wild type after exposure to UVB. Thirteen-day-old
wild type (WT), uvs, and uvt1 plants were exposed to fluorescent
lighting with (⫹) or without (⫺) additional 0.15 W m⫺2 UVBradiation for 21 h. Extracts were prepared from equal amounts of
tissue in 80% (v/v) ethanol. This figure represents an extension of
previously published work (Lois and Buchanan, 1994)
to the accumulation of anthocyanins. To test this, we
analyzed the absorption at the characteristic maxima
at 530 nm of anthocyanins in acidified methanol extracts. This method allows specific quantification of
anthocyanins as opposed to A330, which reflects absorbance by a variety of phenolics including anthocyanins. In Figure 5, we can see a significantly higher
level of 530 nm absorption in uvt1 than in wild-type
controls and uvs. This leads us to conclude that uvt1
accumulates higher levels of anthocyanins in the absence of UV exposure and that this may be the cause,
at least in part, for the darker coloration in the leaves
and petioles of these plants (see Fig. 2).
Because both flavonoids and sinapates absorb in
the UVB range and have been implicated in UV
protection (Landry et al., 1995), we set out to determine if the increased UVB absorption in uvt1 could
be mediated by an increased accumulation of sinapates as well as flavonoids. HPLC analysis of pigment extracts (Fig. 6) showed that several comPlant Physiol. Vol. 126, 2001
pounds (the most prominent peaks in the A330
chromatogram) display significantly elevated levels
in uvt1 when compared with wild-type plants. Fluorescence monitoring of the eluate showed that one of
the major peaks of absorbance (arrow) corresponds
to a highly fluorescent compound that accumulates
at a higher level in uvt1 plants. Chapple et al. (1992)
showed that these highly fluorescent compounds in
Arabidopsis correspond to sinapates. Based on this
and on our observations that extracts from fah1, an
Arabidopsis mutant lacking sinapates, do not display
that prominent peak of fluorescence (data not
shown), we conclude that uvt1 has elevated levels of
one of the sinapates. This is interesting because it
indicates that the effects of the uvt1 lesion involve
two biosynthetic pathways, flavonoid and sinapate,
Figure 5. Anthocyanin levels in mutant and wild-type Arabidopsis
plants. Anthocyanins were extracted in acidified methanol from
leaves of 36-d-old wild-type control (WT) and uvs plants and from
UV-tolerant individuals (uvt1). The height of the bars represents the
mean value of A530 per milligrams tissue of three independent experiments. Error bars correspond to one SD. There is a statistically
significant difference between the uvt1 value and each of the other
two values (non-paired Student’s t test, P ⬍ 0.05).
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1109
Bieza and Lois
Figure 7. Spectra of UV light transmitted through leaves from wildtype and mutant Arabidopsis plants. Irradiance spectra were obtained from incident light (Lamp) and light transmitted through leaves
from 7-week-old wild-type (WT), uvs, and uvt1 plants exposed to
UVB radiation. The left ordinate corresponds to the spectra of UV
transmitted through the leaves and the right ordinate to the incident
light spectrum.
Figure 6. HPLC separation of UV-absorbing compounds from leaf
tissues of uvt1 and wild-type plants. Pigments extracted from 16-dold plants in 70% (v/v) methanol were separated by HPLC on a C18
column. The spectra represent the elution profiles between 9 and 19
min (the region where the most prominent peaks eluted) as monitored by spectrophotometry (A330, thick lines) and fluorescence (thin
lines). The arrow indicates a peak corresponding to a highly fluorescent sinapate.
and suggests that it may affect a common regulator
of these pathways.
Because we detected the accumulation of UVBabsorbing compounds in uvt1 using leaf extracts, the
possibility existed that these pigments may not be
distributed homogeneously in the leaf and thus not
really function as a protective filter for the whole leaf
surface. This is especially true in the case of anthocyanins because of their localized accumulation in
petioles and leaf midrib. If all UV-absorbing compounds were non-homogeneously distributed, their
accumulation would not result in protection to most
of the leaf surface. To address this question, we measured the capacity to absorb UV at different places on
the lamina of different leaves, excluding the midrib
in our mutants and in control plants. More specifically, we determined the amount of UV radiation
transmitted through uvt1 leaves and compared it
with that transmitted by uvs and control wild-type
leaves. Figure 7 shows the spectra of light transmitted through the different types of leaves and Figure 8
shows the level of transmittance of UVB radiation
through these leaves. In both figures, it is clear that
highest and lowest transmission of UVB occurred in
the cases of uvs and uvt1 leaves, respectively,
whereas intermediate transmission was seen in wildtype leaves. Therefore, a one-to-one correlation exists
1110
between high tolerance to UV and leaf absorption of
UVB radiation. Furthermore, despite some variation
in the percent transmission between different places
on the lamina or between different leaves, in all cases
uvt1 leaves transmitted less UVB than either uvs or
wild-type controls. Thus, the distribution of UVabsorbing compounds in these leaves is homogeneous enough for these pigments to filter UV differentially and thus protect the whole leaf area from UV
damage. As a consequence, the tight correlation between in vivo leaf absorption in the UVB range and
the overall resistance of the plant to UVB radiation
Figure 8. Relative transmission of UVB light through wild-type and
mutant Arabidopsis plants. Irradiance spectra were obtained from
incident light (Lamp) and light transmitted through leaves from
7-week-old wild-type (WT), uvs, and uvt1 plants exposed to UVB
radiation. The height of the bars indicate the averages of percent
transmission measurements for wavelengths between 290 and 320
nm taken in three or four places in each leaf lamina from at least
three different leaves from uvs, wild-type, and uvt11 plants. Error
bars correspond to one SD. The numbers above each bar represent the
full range of percent transmittance values for each plant type.
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Plant Physiol. Vol. 126, 2001
Arabidopsis Mutant Hyper-Tolerant to UVB Radiation
suggest an important role for UV-absorbing compounds in the tolerance of uvt1 to UVB. In summary,
uvt1 plants display an extreme tolerance to UV radiation levels that would kill the wild type, which may
be explained by the increased levels of UV-absorbing
compounds deposited in their leaves.
Chalcone Synthase (CHS) mRNA Levels in uvt1 Are
Constitutively Elevated
Thus far, our results show that uvt1 accumulates
higher levels of UV-absorbing compounds in plants
that have not been exposed to the stimulus of UV
radiation. We have shown that some of these compounds are pigmented flavonoids and other phenolics, and that their excessive accumulation is responsible, at least in part, for the darker coloration of uvt1.
As a first step in defining the mechanism underlying
the changes in pigment accumulation, we wanted to
determine if this elevated constitutive accumulation
of phenolics in uvt1 was due to changes in gene
expression. To this end, using RNA-blot analysis, we
measured the mRNA levels of CHS, the gene encoding the enzyme that catalyzes the first committed
step to flavonoid biosynthesis. The results in Figure 9
show a significantly higher level of CHS mRNA accumulation, before exposure to UV, in uvt1 than in
wild type (wild type/⫺UV and uvt1/⫺UV). In fact,
we were unable to detect any CHS transcripts in
wild-type plants not exposed to UV even after a
much longer autoradiographic exposure of the blot
(data not shown). Thus, uvt1 displays elevated levels
of expression of CHS mRNA and accumulation of
flavonoids before exposure to UV. Therefore, it is
likely that the increased level of pigment accumulation observed in these plants is the result of altered
gene expression. The fact that uvt1 plants display
increased accumulation of both flavonoids and sinapates argues against the possibility that the CHS
gene may be the mutated locus in uvt1 and suggests
that the defect may be at a gene involved in the
regulation of more than one biosynthetic pathway.
Because CHS gene expression is regulated by various stimuli, including UV light (Tsukaya et al., 1991;
Fuglevand et al., 1996; Ni et al., 1996; van Eldik et al.,
1997; Hartmann et al., 1998; Kubasek et al., 1998), it
was important to investigate if the constitutive expression of CHS in uvt1 was due to a defect in the
UV-mediated regulation of the CHS gene. The data in
Figure 9 (lane 4) show that CHS gene mRNA levels
are significantly elevated after UVB exposure and,
therefore, that the UVB-mediated regulation of CHS
gene expression is still operating in uvt1. The fact that
the UVB signal transduction pathway can still function argues that a full deregulation of the UV signal
transduction pathway leading to increased CHS expression may not be the problem in uvt1. This indicates that uvt1 may have a partially deregulated UV
signal transduction pathway, or alternatively, that
the genetic lesion in uvt1 is affecting one of the other
signaling pathways that control CHS gene expression. The fact that uvt1 plants show shortened hypocotyls and delayed flowering however suggests that
the uvt1 defect might be in one of the components of
the white light regulation of CHS, perhaps the induction of CHS mediated by high irradiance (Feinbaum
et al., 1991).
As was the case with UV-absorbing pigment accumulation, we also found a tight correlation between
high CHS expression and increased resistance to UV.
Analysis of CHS expression in pools of over 500
plants from the UV-sensitive or the UV-tolerant populations from a selfing cross of a uvt1 heterozygote
resulted in high level of CHS mRNA both before and
after UV irradiation in the UV-resistant population,
whereas undetectable levels were observed in the
UV-sensitive pools (data not shown). Thus, the UVB
tolerance of uvt1 correlates with deregulated gene
expression and may even be a consequence of it.
Comparison of uvt1 with Other Mutants That Show
Altered CHS Expression
Figure 9. Accumulation of CHS mRNA in mutant and wild-type
Arabidopsis. Northern blot from total RNA samples isolated from
aerial tissues of 13-d-old wild-type, uvt11, and uvs plants not exposed (⫺UV) and exposed (⫹UV) to 0.15 W m⫺2 UVB radiation for
21 h, hybridized with a CHS gene probe. The upper image shows the
rRNA stained with methylene blue. This image was digitized before
printing.
Plant Physiol. Vol. 126, 2001
Jackson et al. (1995) described a mutant, icx1, which
like uvt1 shows constitutively elevated CHS gene
expression. The icx1 mutation differs from uvt1, however, in that it segregates as a single recessive lesion
and that it leads to other phenotypic characteristics.
For example, uvt1 does not show the narrower young
leaves or the decreased number of trichomes observed in icx1 leaf. Therefore, it is unlikely for these
two mutations to represent the same genetic locus.
The cop/det/fus family of Arabidopsis mutants has
been described as failing to repress light-activated
genes (Wei and Deng, 1996). One of the characteris-
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1111
Bieza and Lois
tics of the cop, det, and fus mutants is that they display
deregulated expression of CHS and an excessive accumulation of anthocyanins in young seedlings and
in mature seeds. Because the expression of CHS and
anthocyanins levels are also altered in uvt1, we
wanted to know if uvt1 displayed the short hypocotyls of dark grown fusca, det, and cop mutants. Therefore, we grew uvt1, wild-type, and uvs Arabidopsis
seeds in darkness for 12 d and found that uvt1 seedlings displayed an etiolated phenotype, indistinguishable from that of wild-type controls or uvs seedlings (data not shown). We conclude that uvt1 does
not belong to the same class of mutants as the cop/
det/fus mutants and, therefore, that the alteration of
CHS regulation and excessive accumulation of anthocyanins in uvt1 define a new class of mutant with
marked tolerance to UV radiation.
A recently described mutant of Arabidopsis
(pap1-D) generated by activation tagging (Borevitz et
al., 2000) may shed some light on the molecular
mechanisms underlying the uvt1 phenotype. This
mutant overexpresses a MYB transcription factor and
displays elevated pigmentation similar to that in
uvt1. The phenotype of this mutant further resembles
that of uvt1 in that it is caused by a single dominant
lesion that leads to elevated CHS expression as well
as enhanced flavonoid and sinapate accumulation.
These similarities in the phenotypes of both mutants
suggest that both mutations might affect similar if
not identical targets. However, these two mutants
differ from each other in their pigmentation pattern.
The enhanced pigmentation of uvt1 is restricted to
leaves and stems, whereas in pap1-D it affects the
whole plant including roots, sepals, anthers, and carpels in addition to stems and leaves. These differences do not appear to be merely a quantitative difference in pigment accumulation between the two
mutants, but a differential effect of each mutation in
different organs. For example, the level of pigmentation in roots and stems are comparable in pap1-D
plants, whereas no pigmentation is detected in the
roots of uvt1 plants that show deep stem pigmentation. This organ specificity in uvt1 pigmentation and
the lack thereof in pap1-D then argues that these two
mutations might be affecting different regulatory targets. One alternative is that these different targets
may be part of the same gene. Another alternative is
that the uvt1 mutation affects a different regulator of
phenylpropanoid biosynthesis and that this leads to
similar pigmentation effects but with different organ
specificity. Preliminary mapping data of the uvt1
mutation has ruled out a close linkage to pap1-D or
to the related PAP2 gene (L. Procter and R. Lois,
unpublished data) lending support to the latter
alternative.
In conclusion, we have presented evidence that a
dominant mutation in Arabidopsis, leading to altered
expression of the CHS gene and to increases in accumulation of UV-absorbing compounds, confers a
1112
high level of tolerance to UV radiation in these
plants. The uvt1 mutant may prove to be an important tool not just to help define in more exact terms
the role of UV-absorbing pigments in the overall
tolerance to UV radiation but also to help elucidate
the relative contribution of different mechanisms of
protection against UV. For example, we expect that
studies of the combined effects of the uvt1 mutation
in the background of some of the already known
mutations leading to increased UV sensitivity (like
fah1 and the different DNA repair mutants) may help
us understand the way in which different mechanisms of protection interact and how they contribute
to the overall tolerance of a plant to UV radiation.
Finally, we expect that uvt1 in combination with
other pigmentation mutants may help understand
the molecular mechanisms regulating phenylpropanoid biosynthesis in different organs.
MATERIALS AND METHODS
Plant Growth and UVB Treatment
Arabidopsis (Columbia/glabrous-1 mutant) were grown
in potting soil (Home Pro, Chico, CA, or Sunshine Special
Blend, McConkey Co., Sumner, WA) under white fluorescent light (between 90–150 ␮Ei m⫺2 s⫺1 photosynthetically
active radiation) in 12-h-light/-dark cycles at 23°C. For UV
treatments, plants were transferred to continuous lighting
of white fluorescent light (between 30–70 ␮Ei m⫺2 s⫺1
photosynthetically active radiation), supplemented with
0.10 to 0.18 W m⫺2 UVB light (from one fluorescent F40
UVB bulb, Phillips, Holland; or an FS40T12 UVB bulb,
Light Sources, Milford CT). Nonexposed control plants
were either covered with a 0.13-mm mylar filter that blocks
wavelengths of light below 320 nm, or maintained in a
different incubator in the absence of UV under similar
white light conditions.
For dark-growth experiments, seed samples were surface sterilized in a solution of 0.1% (v/v) Triton X-100 and
10% (v/v) bleach in water for 10 min and rinsed three times
in vast excess of sterile double distilled water for 20 min.
Sterilized seeds were sown on 4 g L⫺1 agar containing 2%
(w/v) Suc and Murashige and Skoog basal salts (Gibco Life
Technologies, Inc., Grand Island, NY).
Mutagenesis
Seeds from the Arabidopsis uvs mutant (Lois and
Buchanan, 1994; Columbia/glabrous-1 background), were
exposed to 0.20% (v/v) EMS (Sigma, St. Louis) for 12 to
16 h. Seeds were rinsed twice in vast excess double distilled
water, soaked in double-distilled water for 24 h, and dried.
Approximately 10,000 seeds were sown, grown for 2
weeks, and then exposed to 0.15 W m⫺2 UV radiation for
2 d or until plants showed signs of visible damage. Prospective UV-tolerant mutants (uvt) were selected based on
a lack of visible damage and a healthier overall appearance
compared with the rest of the plants. Selected plants were
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Plant Physiol. Vol. 126, 2001
Arabidopsis Mutant Hyper-Tolerant to UVB Radiation
transplanted, allowed to recover under white fluorescent
light, and self-crossed.
It should be noted that because of the uvs genetic background, we chose Columbia/gl-1 plants (wild type for the
uvs and uvt1 loci) as the most appropriate controls for all
experiments. We refer to these plants as wild-type controls
throughout the paper unless it is specifically stated
otherwise.
Plants from a self-cross of uvt1 showing the UV-tolerant
phenotype (in a uvs and gl1 background) were used as
female recipient for a cross to Columbia plants wild type
for both uvs and gl1. From these populations segregating for
uvt1, gl1,and uvs, we were able to obtain uvt1 plants in a
wild-type genetic background for both the gl1 and uvs loci.
Spectrophotometric Analysis
Pigment extracts were prepared by incubating 0.1 g of
leaf tissue in 500 ␮L 80% (v/v) ethanol at 65°C for 10 to 20
min. Absorption spectra of undiluted extracts were obtained using a diode array spectrophotometer (Beckman
DU 7400, Fullerton, CA). In those cases where the total
aerial tissues were used for pigment extraction, absorbance
values are presented as a ratio between A330 to milligrams
of tissue.
Anthocyanin Analysis
Leaf samples were ground in liquid nitrogen and extracted overnight in 1.0 mL 1% (v/v) HCl, in methanol.
After extraction, 1.0 mL of water and 2.0 mL of chloroform
were added, and the sample was vortexed and centrifuged
at 3,000g for 2 min. Relative anthocyanin levels were determined by measuring A530 of the upper aqueous phase
(Mancinelli et al., 1988) using a Beckman DU 7400
spectrophotometer.
HPLC Analysis
Leaf samples (0.4g) were ground in liquid nitrogen and
extracted in a solution of 1.6 mL 70% (v/v) methanol and
1% (v/v) HCl. The extract was clarified by centrifugation at
5000g for 3 min and filtered through a 0.22-␮m membrane
before injection into a 4.6- ⫻ 250-mm Partisphere RTF C18
column (Whatman, Clifton, NJ). HPLC chromatography
was carried out using a Shimadzu HPLC system with a UV
spectrophotometric detector monitoring A330 and a Kratos
FS970 LC-Fluorometer (Kratos, Chestnut Ridge, NY) using
350-nm excitation and a 370-nm emission cutoff filter.
Chromatography was based on a modification of procedure described by Graham (1991) and Li et al. (1993).
Chromatography essentially was at 1.0 mL min⫺1 using
increasing concentrations of HPLC grade acetonitrile
mixed with 0.01% (v/v) glacial acetic acid in water. After
injection, acetonitrile was increased linearly from 1% to
32% (v/v) in 5 min, maintained at 32% (v/v) for 5 min,
increased linearly to 34% (v/v) in 5 min, to 37% (v/v) in 2
min, to 40% (v/v) in 3 min, and to 99% (v/v) and maintained at 99% (v/v) for 5 min.
Plant Physiol. Vol. 126, 2001
Radiometric Analysis
Irradiance and transmission measurements were performed with a CCD diode array detector (model Instaspec
IV, Oriel, Stratford, CT) connected to a Spectrograph/
Monochromator (Oriel model Multispec 1/8M) with
0.2-nm resolution using a 1,200 lines mm⫺1 grating and a
120-␮m slit. The input to the Monochromator was either a
15.3-cm integrating sphere (Oriel model 70451), for most
irradiance determinations, or a 2-m, small slit, high-grade
fused silica fiber optic cable (Oriel model 77532) for the
measurements of the transmission spectra through intact
leaves. Comparative measurements of UV irradiance were
routinely performed with a hand-held UV light meter with
a UVB probe (Oriel Goldilux model 70215/70221). All three
instruments were calibrated with a standard of spectral
irradiance lamp (Oriel model 63361) following the manufacturer’s instructions. In the transmission experiments, to
avoid differential reflection due to trichomes, all plants
used had a glabrous phenotype.
CHS mRNA Analysis
For each treatment, approximately 0.1 g leaf tissue was
harvested, frozen in liquid nitrogen, and stored at ⫺80°C
until extraction. Frozen tissue was ground using a mortar
and pestle under liquid nitrogen and total RNA was extracted using an RNeasy kit (Qiagen, Santa Clarita, CA).
RNA concentration was determined by A260. Analysis by
RNA blotting was carried out as a modification of the
methods described by Sambrook et al. (1989). For electrophoresis, RNA samples were prepared by mixing 3 ␮L of
RNA with 17 ␮L RNA loading buffer (57% [v/v] formamide, 20% [v/v] formaldehyde, 0.025% [w/v] bromphenol blue, 0.025% [w/v] xylene cyanole FF, 3% [v/v] glycerol, 5 mm NaH2PO4, 5 mm Na2HPO4, 5 mm sodium
acetate, and 1 mm EDTA), incubated at 65°C for 10 min,
then cooled on ice before loading. Five micrograms total
RNA was separated on a 1.2% (w/v) agarose 6% (v/v)
formaldehyde denaturing gel in 1⫻ phosphate running
buffer (5 mm NaH2PO4, 5 mm Na2HPO4, 5 mm sodium
acetate, and 1 mm EDTA). Samples were electrophoresed at
70 V for 3 to 4 h, transferred by capillary elution to a
Gene-Screen nylon membrane (NEN Life Science, Boston)
in 25 mm NaH2PO4 (pH 6.5) for 16 h, cross-linked at 1,200
␮W cm⫺2 with a CL-1000 UV cross-linker (Ultra-Violet
Products, Inc., San Gabriel, CA), and stained with 0.04%
(w/v) methylene blue in 0.5 m sodium acetate for 5 min to
visualize rRNA.
Probe Synthesis
Primers 5⬘-CTGACTACTACTTCCGCATC-3⬘ and 5⬘GTGATCTCAGAGCAGACAAC-3⬘ (Universal DNA, Inc.,
Tigard, OR) were used to amplify a 453-bp fragment from
the CHS gene from Arabidopsis cDNA to use as a probe.
For cDNA synthesis, 1.5 ␮g total leaf RNA, pre-incubated
at 65°C for 10 min and cooled on ice, was incubated for 1 h
at 37°C with 13.3 units Moloney Murine Leukemia Virus
(M–MLV) reverse transcriptase (Promega, Madison, WI);
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1113
Bieza and Lois
1.5 ␮m Oligo dT 15 mer primer; 0.5 ␮m each of dATP,
dCTP, dGTP, and dTTP (Boehringer Mannheim, Indianapolis); 30 units of RNase inhibitor (Stratagene, LaJolla, CA);
and 3 ␮L 10⫻ M-MLV reverse transcriptase buffer (Promega) in a total volume of 30 ␮L. Synthesis of cDNA was
terminated by incubating at 70°C for 10 min and samples
were kept at ⫺20°C until used for probe amplification.
Amplification reactions were carried out using approximately 1 ␮g of cDNA, with 0.5 unit of Taq polymerase
(Promega); 20 mm each of dATP, dCTP, dGTP, and dTTP
(Boehringer Mannheim); 50 ␮m each of CHS primer; 10 ␮L
of 10⫻ Taq polymerase buffer (Promega); and 25 mm magnesium chloride in a 100-␮L reaction. Amplification reactions consisted of 1 cycle at 94°C for 3 min, 30 cycles of 55°C
for 1.5 min, 72°C for 2 min, and 94°C for 2 min, and a final
72°C incubation for 2 min.
Amplified fragments were purified after electrophoresis
on a 1.5% (w/v) agarose gel containing 0.5 ␮g mL⫺1
ethidium bromide (Sigma), and band excision using QIAquick Gel Extraction Kit (Qiagen). A 25-ng sample of amplified CHS probe was labeled using 32P-labeled dCTP by
random priming (Boehringer Mannheim) following the
manufacturer’s recommendations. Unincorporated nucleotides were removed from the radiolabled probe by spinning through a 1-mL syringe filled with Sepharose CL-6B
(Sigma). Prehybridization was carried out in a hybridization oven at 42°C in 15 mL 0.75 m NaCl, 50 mm NaH2PO4,
5 mm EDTA, 50% (v/v) formamide (Fluka Chemical Corp.,
Ronkonkoma, NY), 0.1% (w/v) SDS, 0.5% (w/v) nonfat dry
milk, and 1.7 ␮g mL⫺1 denatured calf thymus DNA for 20
to 30 min. Hybridization was overnight and included 2 ⫻
107 cpm of denatured probe in 15-mL prehybridization
buffer. Membranes were washed three times in approximately 300 mL of 0.3 m NaCl, 20 mm NaH2PO4, 2 mm
EDTA, and 0.1% (w/v) SDS at 60°C for 25 min. The membrane was wrapped with plastic wrap, and autoradiographed with Kodak Imaging film (Blue XB-1) with Kodak
X-Omatic regular intensifying screens from overnight to 3
weeks at ⫺80°C depending on the signal.
ACKNOWLEDGMENT
We thank Min-Young Ahn for excellent technical
assistance.
Received February 15, 2001; returned for revision April 3,
2001; accepted April 19, 2001.
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