Redox-Dependent Modulation of Anthocyanin
Biosynthesis by the TCP Transcription
Factor TCP15 during Exposure to High Light Intensity
Conditions in Arabidopsis1[OPEN]
Ivana L. Viola, Alejandra Camoirano, and Daniel H. Gonzalez*
Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Cátedra de Biología Celular y Molecular, Facultad
de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina
ORCID ID: 0000-0002-9929-287X (A.C.).
TCP proteins integrate a family of transcription factors involved in the regulation of developmental processes and hormone
responses. It has been shown that most members of class I, one of the two classes in which the TCP family is divided, contain a
conserved Cys that leads to inhibition of DNA binding when oxidized. In this work, we describe that the class-I TCP protein
TCP15 inhibits anthocyanin accumulation during exposure of plants to high light intensity by modulating the expression of
transcription factors involved in the induction of anthocyanin biosynthesis genes, as suggested by the study of plants that
express TCP15 from the 35SCaMV promoter and mutants in TCP15 and the related gene TCP14. In addition, the effect of TCP15
on anthocyanin accumulation is lost after prolonged incubation under high light intensity conditions. We provide evidence that
this is due to inactivation of TCP15 by oxidation of Cys-20 of the TCP domain. Thus, redox modulation of TCP15 activity in vivo
by high light intensity may serve to adjust anthocyanin accumulation to the duration of exposure to high irradiation conditions.
TCP proteins constitute a family of plant-specific
transcription factors involved in the regulation of cell
proliferation and growth-related processes. TCP transcription factors contain a conserved DNA binding and
dimerization domain, the TCP domain, that bears a
resemblance to the bHLH domain found in many eukaryotic transcription factors (Cubas et al., 1999). Arabidopsis contains 24 TCP proteins that can be ascribed
to classes I or II according to sequence conservation
within the TCP domain (Cubas et al., 1999; MartínTrillo and Cubas, 2010). Class-I proteins participate in
the regulation of cell proliferation, leaf and flower development, stem elongation, and responses to hormones.
They modulate jasmonic acid biosynthesis and cytokinin, auxin, and gibberellin responses (Martín-Trillo and
1
This work was supported by grants from the Agencia Nacional
de Promoción Científica y Tecnológica, Argentina, the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and the
Universidad Nacional del Litoral. D.H.G. and I.L.V. are members of
CONICET. A.C. is a CONICET fellow.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Daniel H. Gonzalez ([email protected]).
I.L.V. and D.H.G. designed the experiments and analyzed data.
I.L.V. performed the experiments. A.C. performed and analyzed
RT-qPCR measurements. D.H.G. wrote the manuscript. I.L.V. made
the figures and contributed to writing.
[OPEN]
This article is available without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.01016
74
Cubas, 2010; Manassero et al., 2013). Mutations in genes
encoding two closely related class-I proteins from Arabidopsis, TCP14 (At3g47620) and TCP15 (At1g69690),
affect several aspects of plant development, among them
seed germination (Resentini et al., 2015), leaf shape
(Kieffer et al., 2011), inflorescence stem growth (Davière
et al., 2014), and gynoecium development (Lucero et al.,
2015). Recently, it was also shown that mutations in these
genes affect effector-triggered immunity (Kim et al.,
2014), thus suggesting a role of class-I TCP proteins in
responses to stress.
As part of the sequence conservation observed within
the TCP domain of class-I proteins, we have previously
identified a conserved Cys located at position 20 of
the TCP domain. We have shown that this Cys undergoes redox interconversions that render the proteins
unable to bind DNA when oxidized (Viola et al., 2013).
According to this, we have postulated that class-I TCP
proteins may participate in responses to internal or
external conditions that cause changes in cell redox
parameters. Changes in the production or amounts of
redox compounds, mainly due to the generation of reactive oxygen species (ROS), are observed under a variety of stress conditions (Noctor and Foyer, 1998; Grant
and Loake, 2000; Foyer and Noctor, 2005; Potters et al.,
2009; Frederickson Matika and Loake, 2014). However,
little is known about the participation of class-I TCP
proteins in responses to stress.
A stress condition that causes changes in redox parameters is the growth of plants under high light intensities. Excess light causes changes in the redox state
of photosynthetic electron transport chain components
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Redox Modulation of TCP15 by High Light Intensity
and thiol compounds and increases ROS production
(Karpinski et al., 1997; Huner et al., 1998; Foyer and
Allen, 2003; Li et al., 2009). These changes originate
protective responses oriented to prevent cell damage
under the new situation. A typical response to high
light intensities is the accumulation of anthocyanins,
a group of aromatic pigments that have protecting
functions against excess light and ROS (Holton and
Cornish, 1995; Grotewold, 2006; Albert et al., 2009;
Agati et al., 2012; Page et al., 2012). Anthocyanins act as
protecting pigments since they absorb light and thus
reduce the amount of energy that reaches the photosynthetic apparatus (Hughes et al., 2005; Albert et al.,
2009). It has been postulated that anthocyanins also act
as antioxidant compounds in plants since they are ROS
scavengers (Neill et al., 2002; Nakabayashi et al., 2014).
Anthocyanin synthesis is modulated at the transcriptional level through the regulation of anthocyanin
biosynthesis genes (i.e. genes that encode enzymes involved in anthocyanin biosynthesis) by transcription
factors from the Myb, bHLH, and WD40 families
(Winkel, 2006; Guo et al., 2014). These factors form a
transcriptional complex, called MBW, which binds to
anthocyanin biosynthesis gene promoters and induces
gene expression (Quattrochio et al., 2006). Some members of the MBW complex are in turn also regulated by
light conditions at the transcriptional and posttranslational levels (Lee et al., 2007; Shin et al., 2007; Dubos
et al., 2008; Matsui et al., 2008; Maier et al., 2013; Albert
et al., 2014). It was recently shown that the class-II TCP
protein TCP3 induces anthocyanin biosynthesis by integrating into the MBW complex (Li and Zachgo, 2013).
In this work, we report that the class-I TCP protein
TCP15 from Arabidopsis acts as a repressor of anthocyanin accumulation acting on the expression of anthocyanin biosynthesis genes and upstream transcriptional
regulators. TCP15 prevents accumulation of anthocyanin under high light intensity after relatively short irradiation times but not after prolonged irradiation. We
provide evidence indicating that loss of TCP15 action
after prolonged irradiation is due to inactivation of
TCP15 by oxidation of Cys-20 of the TCP domain. Redox
interconversions of TCP15 and other class-I proteins
would then help to modulate the response of plants to
high light intensities depending on the duration of these
external conditions.
leaves, petioles, and inflorescences (Uberti-Manassero
et al., 2012), which is in agreement with expression data
from microarray experiments (http://bar.utoronto.ca/).
Upon growing these plants under standard conditions,
as described in “Materials and Methods”, we observed
typical signs of anthocyanin accumulation. Analysis of
global gene expression changes in rosettes of these plants
revealed the induction of 740 genes. Among them, several genes involved in anthocyanin biosynthesis were
observed (Fig. 1; Supplemental Table S1). In fact, 42% (15
out of 36) of the anthocyanin biosynthesis or regulatory
genes described by Guo et al. (2014) are induced in p15:
TCP15-EAR plants, which is significantly higher than
expected by chance (P , 0.001). Four of these genes encode transcription factors involved in the positive regulation of the expression of anthocyanin biosynthesis
genes (PAP1, PAP2, TT8, and EGL3) and 11 encode enzymes involved in different steps of anthocyanin biosynthesis from chalcones (Fig. 1). In addition, TT19, a
gene encoding a transporter involved in the accumulation of anthocyanins in the vacuole (Kitamura et al.,
2004; Sun et al., 2012), and AT5MAT, which encodes
an anthocyanin 5-O-glucoside-699-O-malonyltransferase
involved in the malonylation of anthocyanin (D’Auria
et al., 2007), are also induced in p15:TCP15-EAR plants
(Fig. 1). Notably, genes encoding enzymes involved in
the synthesis of isoflavonoids, flavonols, and proanthocyanidins, which share metabolic precursors with anthocyanins, did not show changes in expression or were
repressed, as in the case of the flavonol biosynthetic
genes FLS2 (Owens et al., 2008; Guo et al., 2014),
UGT78D1 (Jones et al., 2003), and UGT71D1 (Lim et al.,
2004) (Supplemental Table S1).
Accumulation of anthocyanins in p15:TCP15-EAR
plants was evident after visual inspection (Fig. 2 A, arrowheads). Measurement of anthocyanin levels indicated
that, indeed, p15:TCP15-EAR plants accumulate higher
anthocyanin levels than wild-type plants (Fig. 2 B). We
also measured transcript levels of TT8 and DFR, anthocyanin regulatory and biosynthesis genes, respectively
(Fig. 1), by RT-qPCR. The results confirmed the induction
of these genes in p15:TCP15-EAR plants (Fig. 2 C) and
suggest that TCP15 may be involved in the modulation of
anthocyanin levels through the regulation of the expression of anthocyanin biosynthesis and regulatory genes.
TCP15 Is a Negative Regulator of Anthocyanin
Accumulation under High Light Conditions
RESULTS
Expression of a Repressor Form of TCP15 Causes
an Increase in Anthocyanin Levels
Previously, we described plants that express a fusion
of the Arabidopsis TCP transcription factor TCP15
to the EAR repression domain under the control of
the TCP15 promoter (p15:TCP15-EAR plants) (UbertiManassero et al., 2012). These plants express TCP15EAR from a 1514-bp fragment containing sequences
located upstream of the TCP15 translation start codon.
This fragment promotes expression in developing
To further evaluate the role of TCP15 in anthocyanin accumulation, we analyzed anthocyanin levels in
plants that express TCP15 from the 35SCaMV promoter
(35S:TCP15 plants) and in double knock-out mutants in
TCP15 and the related TCP protein TCP14. We used the
double knock-out mutant with TCP14 for our studies,
since it has been shown that there is a high degree of
redundancy in the functions of TCP15 and TCP14
(Kieffer et al., 2011; Davière et al., 2014). No differences
in anthocyanin levels were observed among mutant,
wild-type, and overexpressing plants under standard
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Figure 1. Genes involved in anthocyanin production are induced in p15:TCP15-EAR plants. A, Scheme of the anthocyanin
biosynthesis pathway in Arabidopsis. The abbreviated names of the genes encoding enzymes involved in anthocyanin biosynthesis are shown, together with the names of transcription factors involved in the induction of anthocyanin biosynthesis genes.
Names boxed in gray indicate genes whose expression is increased in p15:TCP15-EAR plants. B, List of genes involved in anthocyanin accumulation whose expression is induced in p15:TCP15-EAR plants grown under standard light conditions. logFC is
the log2 of the fold-change in expression in p15:TCP15-EAR plants relative to wild-type (wt) plants. Details of gene names and
AGI codes are given in Supplemental Table S1.
conditions (Fig. 3 A). We then decided to apply a
treatment that induces anthocyanin accumulation to
analyze the response of plants in which the function of
TCP15 was altered. For this purpose, we treated plants
with high light intensity during the light period (see
“Materials and Methods” for details) and analyzed
anthocyanin levels at different times after the beginning
of the treatment. At d 3, wild-type plants showed a
2.5-fold increase in anthocyanin levels (Fig. 3 A). These
levels were similar to those observed in p15:TCP15EAR plants either before or after the high light intensity
treatment (Supplemental Figure S1). In two different lines
of 35S:TCP15 plants, however, anthocyanin levels were
significantly lower than in wild-type plants at d 3 of
treatment, when tcp14 tcp15 mutants contained higher
anthocyanin levels than wild-type plants (Fig. 3 A).
These results suggest that TCP15 is a negative regulator
of anthocyanin accumulation. Notably, anthocyanin levels
were similar in all plants when analyzed at d 5 (Fig. 3 A).
We then measured the response of genes involved in
the regulation of anthocyanin biosynthesis (PAP1, TT8)
and the anthocyanin biosynthesis gene DFR to high
light treatment. As observed in Figure 3 B, tcp14 tcp15
plants showed higher transcript levels of the three
genes after 1 h and 2 h of treatment, indicating that
mutant plants respond more rapidly to high light conditions. For TT8 and DFR, transcript levels were already higher in tcp14 tcp15 plants before the treatment
was started (Fig. 3 B). The increased response of anthocyanin regulatory and biosynthesis genes observed
in tcp14 tcp15 plants may explain why these plants accumulate higher anthocyanin levels than wild-type. In
the case of 35S:TCP15 plants, the opposite behavior was
observed since transcript levels for PAP1, TT8, and DFR
were significantly lower than those of wild-type plants
after 3 h of incubation under high light intensity (Fig. 3 C).
Prolonged High Light Treatment Abolishes the Effects of
TCP15 Overexpression
It is noteworthy that the effect of modifying TCP15
function on anthocyanin accumulation under high light
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Redox Modulation of TCP15 by High Light Intensity
Figure 2. p15:TCP15-EAR plants contain higher anthocyanin levels. A, A photograph of wild-type (wt) and three different lines of
p15:TCP15-EAR plants showing accumulation of anthocyanin in leaves (arrowheads). B, Anthocyanin levels in extracts from
rosettes of wt and p15:TCP15-EAR plants. Note that the higher anthocyanin content is readily evident by visual inspection of the
extracts after chlorophyll extraction, as indicated by the photographs to the right (top: wt; bottom: p15:TCP15-EAR). The bars
indicate the mean 6 SD of three independent measurements. Columns with different letters are significantly different at P , 0.05
(Student’s t test). C, Relative transcript levels of TT8 and DFR in wt and p15:TCP15-EAR plants measured by RT-qPCR. The value in
wild-type plants was set to 1. The bars indicate the mean 6 SD of three technical replicates. Columns with different letters are
significantly different at P , 0.05 (Student’s t test). The experiment was repeated three times with similar results.
was lost after five days of treatment (Fig. 3 A). One
possibility is that TCP15 regulates the speed of the response to high light, but eventually anthocyanin levels
reach a maximum that is similar regardless of the
action of TCP15. Another possibility is that a TCP15independent pathway becomes prevalent after prolonged high light treatment. Finally, a third possibility
is that TCP15 action is inhibited by prolonged exposure to high light. To analyze this, we evaluated the
effect of high light irradiation on the action of TCP15
in a supposedly different pathway, i.e. regulation of
auxin homeostasis. In fact, it has been reported previously that TCP15 regulates auxin homeostasis and
that expression of TCP15-EAR induces the expression
of the auxin reporter DR5:GUS (Uberti-Manassero
et al., 2012). We then crossed DR5:GUS plants with 35S:
TCP15 plants and analyzed the effect of high light
treatment on DR5:GUS expression. As expected from
the results reported for TCP15-EAR, 35S:TCP15 plants
showed significantly lower DR5:GUS expression levels
than wild-type under normal illumination conditions
(Fig. 4 A, left panels). When wild-type plants were
kept under high light intensity, a progressive decay in
DR5:GUS expression was evident (Fig. 4 A, top panels).
This may be related to the fact that auxin responses are
attenuated under stress conditions that generate ROS
(Blomster et al., 2011; Tognetti et al., 2012; Peer et al.,
2013). In turn, treatment of DR5:GUS x 35S:TCP15
plants caused a completely different response, since a
significant increase in DR5:GUS expression was observed after prolonged high light treatment (Fig. 4 A,
bottom panels). Repression of anthocyanin accumulation by 35S:TCP15 followed a similar pattern (Fig. 4 B),
even if the treatment produced opposite behaviors of
both processes in wild-type plants (i.e. increase in anthocyanin accumulation and decrease in DR5:GUS expression). This supports the idea that TCP15 is
inactivated after prolonged high light irradiation, thus
relieving the repression of DR5:GUS expression and
anthocyanin accumulation.
A C20S Mutant of TCP15 Causes Durable Repression of
Anthocyanin Accumulation under High Light Intensity
One of the effects of high light intensity is the accumulation of ROS (Miller et al., 2009). ROS damage cellular structures, cause changes in redox conditions, and
act as intermediates in signal transduction pathways
involved in plant acclimation. We have previously
reported that TCP15 and other class-I TCP proteins
undergo redox interconversions that involve a conserved Cys (Cys-20 of the TCP domain) located at the
beginning of the HLH motif. Oxidation of this Cys inhibits DNA binding and can be achieved in vivo by
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Viola et al.
Figure 3. TCP15 inhibits anthocyanin accumulation during exposure of plants to high light intensity. A, Anthocyanin levels in
wild-type plants (wt), tcp14 tcp15 loss-of-function mutants, and 35S:TCP15 plants (two independent lines) at different times after
exposure to high light irradiation. Irradiation was started at the middle of the light period of d 1. Samples were collected at the end
of the light period of d 3 and d 5. Control samples (d 0) were collected before the treatment was started. No increase in anthocyanin accumulation was observed in plants kept under normal illumination conditions during the treatment. The bars indicate the mean 6 SD of three independent measurements. Columns with different letters are significantly different at P , 0.05
(ANOVA; Tukey test). B, Relative transcript levels of PAP1, TT8, and DFR in wild-type (wt) plants and tcp14 tcp15 mutants after
exposure to high light irradiation. The bars indicate the mean 6 SD of three biological replicates. Columns with different letters are
significantly different at P , 0.05 (ANOVA; Tukey test). C, Relative transcript levels of PAP1, TT8, and DFR in wild-type (wt) and
35S:TCP15 plants (two independent lines) after 3 h of exposure to high light irradiation. The bars indicate the mean 6 SD of three
biological replicates. Asterisks indicate significant differences compared to wt (P , 0.05; ANOVA).
treatment of plants with H2O2 (Viola et al., 2013). We
then asked whether oxidation of Cys-20 of the TCP
domain may be the cause of the apparent inactivation of
TCP15 after prolonged incubation under high light
conditions. To analyze the role of Cys-20 in TCP15
action, we expressed in plants a constitutively active,
redox-insensitive variant of TCP15 obtained by changing Cys-20 to Ser (C20S-TCP15). This replacement does
not affect the DNA binding activity of TCP15 under reducing conditions (Viola et al., 2013). TCP15 transcript
levels in lines that overexpress C20S-TCP15 were similar
to those observed in plants that overexpress native
TCP15 (Supplemental Figure S2). As shown in Figure 5 A,
expression of C20S-TCP15 under the control of the
35SCaMV promoter caused a decrease in anthocyanin
accumulation under high light conditions. At d 2 of
treatment, the effect of C20S-TCP15 was similar to the
one observed with native TCP15 (Fig. 5 A). However,
after more prolonged incubation, repression of anthocyanin accumulation was maintained in 35S:C20S-TCP15
plants but was lost in 35S:TCP15 plants (Fig. 5, A and
B). Similar results were obtained when different lines
expressing either TCP15 or C20S-TCP15 were used (Fig.
5 C), indicating that this is not a line-specific effect. Repression of the expression of TT8 and DFR was also
maintained after prolonged incubation under high light in
35S:C20S-TCP15 plants but not in 35S:TCP15 plants (Fig.
5 D), in agreement with the results of anthocyanin accumulation. The most likely explanation for this observation
is that TCP15 becomes inactivated by oxidation of Cys-20
after prolonged exposure of plants to high light intensities.
TCP15 Is Inactivated after Exposure of Plants to
High light Intensity
To effectively analyze whether TCP15 is inactivated
after exposure of plants to high light, we analyzed the
DNA binding activity of protein extracts prepared from
35S:TCP15 plants toward a double-stranded oligonucleotide that contains the sequence recognized by TCP15
using electrophoretic mobility shift assays (EMSAs)
(Viola et al., 2011). In extracts from nontransformed
plants, two to three faint retarded bands, probably arising
from the binding of endogenous proteins, were observed
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Redox Modulation of TCP15 by High Light Intensity
Figure 4. Repression of DR5:GUS by TCP15 is relieved after prolonged
exposure to high light irradiation. A, Plants carrying the auxin reporter DR:GUS in a wild-type (DR5:GUS) or a 35S:TCP15 background
(DR5:GUS x 35S:TCP15) were exposed to high light irradiation conditions. GUS activity was analyzed by histochemical staining at the beginning of the treatment (day 0) or at the end of the light period of days 2
and 7. B, Anthocyanin levels in the plants treated as described in (A).
Wild-type plants (wt) were also included in the assay for comparison. The
bars indicate the mean 6 SD of three independent measurements. Columns with different letters are significantly different at P , 0.05 (ANOVA;
Tukey test). The experiment was repeated three times with similar results.
(Fig. 6 A). The pattern is similar to the one observed earlier
using seedling extracts and the same oligonucleotide
(Viola et al., 2013). A significant increase in the intensity of
one of the retarded bands was observed when extracts
from 35S:TCP15 plants were used, suggesting that this
represents the binding of TCP15 to the labeled oligonucleotide (Fig. 6 A, asterisk). The specificity of binding to the
TCP15 target site was analyzed in a competition experiment (Fig. 6 B). Binding of 35S:TCP15 extracts was efficiently competed by a 30-fold molar excess of the same,
unlabeled oligonucleotide, but not by a similar amount of
an oligonucleotide carrying two point mutations in the
TCP15 binding site (Fig. 6 B). This strongly suggests that
this retarded band arises from TCP15 binding. In wildtype extracts, no significant competition was observed.
This indicates that the faint retarded bands observed in
wild-type extracts are most likely the consequence of unspecific protein-DNA interactions.
We then performed EMSAs using extracts from
plants that were kept under high light conditions for 2
or 4 days. As observed in Figure 6 C (left panel), binding
of TCP15 was almost completely abolished under these
conditions. However, binding was not affected by a
high light treatment when extracts from 35S:C20S-TCP15
plants were used (Fig. 6 C, right panel). This may indicate
that TCP15 is inactivated by oxidation after high light
treatment of plants. To evaluate this, we treated the
extracts with a reducing agent, since this treatment was
shown previously to reactivate the oxidized protein
(Viola et al., 2013). DTT, as used before in seedling extracts (Viola et al., 2013), was unsuccessful, since incubation in the presence of DTT caused a complete loss of
binding in all samples. We speculate that DTT may
activate a protease or another factor from the extract
that inhibits binding of proteins to DNA. We then used
2-mercaptoethanol (2ME) as reducing agent and observed that the DNA binding activity in extracts from
35S:TCP15 plants subjected to high light intensity conditions was recovered after 2ME treatment (Fig. 6 D,
right panel). The results indicate that TCP15 is present
in an inactive form in extracts from 35S:TCP15 plants
after the high light intensity treatment and that this
form can be reactivated by a reducing agent.
We also analyzed the effect of high light intensity on
the stability of TCP15. For this purpose, we obtained
plants that constitutively express a fusion of TCP15 to
the red fluorescent protein (RFP) and analyzed protein
levels using antibodies against RFP. Figure 7 A shows
that these antibodies detected more than one band in
35S:TCP15-RFP plants but not in nontransformed plants.
This indicates the presence of truncated products, probably arising from proteolytic processing since the RFP tag
is located at the C-terminal end. The band of slowest
migration (Fig. 7 A, arrow) corresponds to the expected
Mr of the full-length protein. TCP15-RFP inhibited anthocyanin accumulation under high light at d 3 of treatment but not at d 5 (Fig. 7 B). This behavior is similar to
the one observed with native TCP15. Analysis of protein
levels showed that the amount of TCP15-RFP did not
change significantly after five days of treatment (Fig. 7 A),
indicating that lack of repression is not due to the disappearance of the protein.
DISCUSSION
TCP15 Is a Repressor of Anthocyanin Accumulation
Plants accumulate anthocyanins in response to several stresses and metabolic conditions (Kubasek et al.,
1992; Batschauer et al., 1996; Winkel-Shirley, 2002;
Lepiniec et al., 2006). One of the functions of anthocyanins is to act as protective pigments to prevent cell
damage under conditions of high light intensity; they
may also act as antioxidants under situations that cause
an increase in ROS accumulation (Grotewold, 2006;
Albert et al., 2009; Agati et al., 2012; Page et al., 2012).
Expression of a repressor form of TCP15 (TCP15-EAR)
in the TCP15 expression domain (i.e. under the TCP15
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Viola et al.
Figure 5. Expression of a C20S-TCP15 mutant causes durable repression of anthocyanin accumulation under high irradiation
conditions. A, Anthocyanin levels in wild-type (wt), 35S:TCP15 (two independent lines), and 35S:C20S-TCP15 plants at different
times after exposure to high light irradiation. The bars indicate the mean 6 SD of three independent measurements. B, Anthocyanin levels in wt and three independent lines of 35S:TCP15 and 35S:C20S-TCP15 plants at d 7 of exposure to high light
irradiation. The bars indicate the mean 6 SD of three independent measurements. C, Photograph of wt, 35S:TCP15, and 35S:
C20S-TCP15 plants at d 7 of irradiation showing the decrease in anthocyanin accumulation in 35S:C20S-TCP15 plants. D,
Relative transcript levels of TT8 and DFR in wild-type (wt), 35S:TCP15, and 35S:C20S-TCP15 plants at different times after exposure to high light irradiation. The bars indicate the mean 6 SD of three biological replicates. Columns with different letters are
significantly different at P , 0.05 (ANOVA; Tukey test).
promoter) causes an increase in anthocyanin levels and
in the expression of several anthocyanin biosynthesis
genes, suggesting that TCP15 modulates anthocyanin
accumulation. This is further supported by the fact that
a tcp14 tcp15 mutant accumulates higher anthocyanin
levels after exposure to high light intensity. This and the
results obtained with plants that express TCP15 from
the 35SCaMV promoter indicate that TCP15 is a repressor of anthocyanin accumulation. The fact that
anthocyanin levels were similar to wild-type in tcp14
tcp15 mutants under normal illumination suggests that
the role of TCP15 and the related protein TCP14 is especially important under conditions that promote anthocyanin accumulation, like the high light intensity
treatment used here. Since p15:TCP15-EAR plants have
higher anthocyanin levels already under normal conditions, this may indicate the existence of redundancy,
probably with other class-I TCP proteins, in the repression of anthocyanin biosynthesis.
Anthocyanin biosynthesis is modulated at the transcriptional level by several transcription factors from the
Myb, bHLH, and WD40 families (Quattrochio et al.,
2006). These factors form the so-called MBW complex
that induces the expression of genes encoding different
enzymes of the anthocyanin biosynthesis pathway. The
genes encoding several of these transcription factors are
also induced under conditions that promote anthocyanin
biosynthesis, suggesting the existence of upstream components in the signaling pathway. Since several of these
genes, such as PAP1, PAP2, TT8, and EGL3, were induced in p15:TCP15-EAR plants, it can be postulated that
TCP15 is one of these upstream components. In addition,
the fact that a repressor form of TCP15 induces the expression of these genes further suggests that the regulation exerted by TCP15 is indirect and probably acts by
inducing the expression of a repressor of PAP1 and other
anthocyanin regulatory genes. Known repressors of anthocyanin biosynthesis include AtMYBL2 (Dubos et al.,
2008; Matsui et al., 2008), CPC (Zhu et al., 2009), SPL9
(Gou et al., 2011), AtMYB4 (Jin et al., 2000; Fornalé et al.,
2014), and AtMYB7 (Fornalé et al., 2014). However, the
genes encoding these transcription factors did not show
changes in expression in p15:TCP15-EAR plants according to the microarray experiment.
It was previously reported that TCP3, a class-II TCP
protein, also regulates anthocyanin biosynthesis. In this
case, TCP3 acts as an inducer of anthocyanin biosynthesis genes by incorporating into the MBW complex
(Li and Zachgo, 2013). This fits into an earlier proposition that class-I and class-II TCP proteins have antagonistic functions (Li et al., 2005). Opposing functions
of class-I and class-II TCP proteins have been shown
for the regulation of senescence and leaf development
associated with jasmonic acid biosynthesis and also
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Redox Modulation of TCP15 by High Light Intensity
Figure 6. High light intensity inhibits
the DNA binding activity of TCP15 in
a redox-dependent manner. A, EMSA
showing the binding of proteins to DNA
carrying a class-I TCP target site. Extracts
from wild-type (wt) and two independent lines of 35S:TCP15 plants were
used. The retarded band highlighted
with an asterisk indicates binding of the
expressed TCP15 to DNA. B, EMSA as in
(A) showing the competition of binding
by a 30-fold molar excess of unlabeled
oligonucleotides carrying an unmodified
(BS) or a mutated (BSm) target site. C,
EMSA using extracts prepared from 35S:
TCP15 and 35S:C20S-TCP15 plants exposed for different times to high light
irradiation. D, EMSA using extracts from
35S:TCP15 and 35S:C20S-TCP15 plants
exposed for 0 or 2 d to high light irradiation. Before loading in the EMSA, the
samples were incubated for 10 min at
25˚C either in the absence (22ME) or presence (+2ME) of 10 mM 2-mercaptoethanol.
The experiment was repeated twice with
similar results.
suggested for the regulation of cell proliferation
(Schommer et al., 2008; Sarvepalli and Nath, 2011;
Danisman et al., 2012). The initial proposition of antagonistic functions was made based on the similarities
of class-I and class-II TCP recognition sequences and
assumed that they were the result of interactions with
the same target sites in gene promoters (Kosugi and
Ohashi, 2002). Although it is highly probable that this
occurs, examples of interactions of class-I and class-II
TCPs with the same target sites are scarce. In the case of
jasmonic acid biosynthesis, class-I and class-II TCPs do
interact with the same target gene (LOX2) but through
different promoter elements (Danisman et al., 2012). In
addition, it is not completely clear that these interactions always lead to antagonistic effects. In the case of
anthocyanin biosynthesis, it has been described that
TCP3 interacts with components of the MBW complex
and strengthens the activation capacity of Myb proteins
bound to this complex (Li and Zachgo, 2013). It would
be interesting to analyze whether TCP15 is capable of
interacting with members of the MBW complex producing the opposite effect, thus inhibiting anthocyanin biosynthesis. Even if this occurs, our results suggest
that the mechanism involved in TCP15 action is
Figure 7. High light intensity does not affect the stability of TCP15. A, Western blot of extracts from wild-type plants (wt) and
plants that express a fusion of TCP15 to RFP (35S:TCP15-RFP). The plants were either maintained under control or high light
irradiation conditions for five days. Blots were revealed with an antibody against RFP. The left and right panels show images of a
Coomassie Brilliant Blue stained gel and a Western blot, respectively, obtained using the same amounts of extracts. The rightmost
lane in the left panel shows molecular-weight standards. The arrow in the right panel indicates the expected migration of fulllength 35S:TCP15-RFP. B, Anthocyanin levels in wild-type (wt) and 35S:TCP15-RFP plants at different times after exposure to
high light irradiation. The bars indicate the mean 6 SD of three independent measurements. Columns with different letters are
significantly different at P , 0.05 (ANOVA; Tukey test).
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Viola et al.
different. (1) An effect on transcription through incorporation of TCP15 to the MBW complex would lead to
repression by TCP15-EAR and activation by TCP15,
which is opposite to a role of TCP15 in inhibiting anthocyanin accumulation, as observed here. (2) A role of
TCP15 in interfering or destabilizing the MBW complex
or the interaction of the complex with TCP3 through
protein-protein interactions would lead to a decrease in
anthocyanin levels independently of the TCP15 form,
either native TCP15 or TCP15-EAR, that is used. The
fact that opposing results are obtained when native or
repressor forms are expressed strongly suggests that
TCP15 acts by directly modulating the expression of
one or more factors that negatively regulate anthocyanin accumulation. However, the primary TCP15 target
genes that relate TCP15 action to anthocyanin biosynthesis remain unknown.
TCP15 Is Inactivated by Oxidation under High
Light Conditions
It seemed intriguing that the action of TCP15 on anthocyanin accumulation was lost after prolonged exposure of plants to high light intensity. However, as
shown under Results, a persistent inhibition of anthocyanin biosynthesis was observed when a redoxinsensitive form of TCP15 was expressed in plants. This
form, C20S-TCP15, carries a mutation in a conserved
Cys present at the beginning of the HLH motif. It has
been reported that oxidation of this Cys inhibits binding of TCP15 to DNA (Viola et al., 2013). Oxidation of
Cys-20, located near the DNA binding domain, may
disrupt DNA binding due to the formation of intermolecular disulfide bonds between two TCP15 monomers (Viola et al., 2013). This is suggested by the fact
that covalent dimers were observed in nonreducing
sodium dodecyl sulfate-polyacrylamide gel electrophoresis recombinant proteins treated with oxidizing agents. In addition, Cys-20 from both monomers are
probably located at a short distance in dimers according
to models of the TCP domain based on its resemblance
with the bHLH domain (Viola et al., 2013). Formation of
covalent dimers may impose spatial constraints that
impair binding of both TCP15 monomers to DNA. It is
also possible that oxidation of Cys-20 to sulphenic or
sulphinic acid, which is also reversible, may impair
DNA binding. Experiments using the two-hybrid system in yeast have shown that the interaction between
TCP15 monomers is not disrupted after H2O2 treatment
(our unpublished data). Mass spectrometry analysis of
TCP15 after inactivation is required to ascertain the
nature of the modification that occurs in vivo.
The results presented in this work indicate that oxidation of Cys-20 occurs after prolonged exposure of
plants to high light intensity. In agreement with this,
binding to DNA was lost in extracts from 35S:TCP15
plants exposed to high light and was recovered after
treatment of the extracts with a reducing agent, suggesting that TCP15 is present in the extract in its oxidized, inactive form. Indeed, binding was not affected
by the high light intensity treatment when C20S-TCP15
was expressed in plants. It has been shown that excess
light causes changes in the pools of redox compounds
and increase the production of ROS, such as H2O2 and
singlet oxygen (Li et al., 2009). Treatment of leaves with
H2O2 was shown previously to inactivate endogenous
TCP15 activity (Viola et al., 2013). Progressive inactivation of TCP15 after exposure to high light may then
be caused by changes in redox conditions of the cellular
environment.
Even if excess light increases ROS production and
promotes anthocyanin accumulation, current evidence
does not support the existence of a direct role of ROS in
the induction of anthocyanin biosynthesis genes. Contrary to that, Vanderauwera et al. (2005) reported that
the induction of anthocyanin biosynthesis genes by
high light is impaired in peroxisomal catalase-deficient
plants, which accumulate high levels of H2O2. These
results may imply that ROS produced during exposure
to high light intensity repress, rather than induce, the
expression of anthocyanin biosynthesis genes, while
our model suggests that ROS act to enhance anthocyanin accumulation after prolonged exposure due to
inactivation of TCP15 and related proteins. One possibility is that the effect of ROS depends on the location or
the species involved. High light intensity causes redox
changes mainly in the chloroplast (Li et al., 2009) and
this may affect redox balance in other compartments in
a different way than H2O2 from peroxisomes. In addition, catalase-deficient plants are constitutively stressed
and this may affect a proper response to ROS produced
during the high light intensity treatment.
It is noteworthy that inactivation of TCP15 by high
light was observed already at d 2, while differences in
anthocyanin accumulation persisted until d 3. Gene
expression changes, on the other hand, showed correlation with TCP15 activity, since differences with wildtype were lost at d 2. This indicates that, once TCP15 is
inactivated, a certain time is required for plants to reach
similar anthocyanin levels than wild-type. It should be
kept in mind that the treatment applied consists of illumination under high light intensity only during the
light period (8 h) and that some recovery may occur
during the night. In any case, the results show that
TCP15 inactivation precedes anthocyanin accumulation, which is consistent with a role of TCP15 redox
changes in modulating anthocyanin levels under high
light conditions. The fact that differences in anthocyanin accumulation in tcp14 tcp15 mutants were also
evident at d 3 but not at d 5 is also consistent with a role
of TCP15 and TCP14 only after relatively short exposure times. After prolonged treatment, inactivation of
the proteins in wild-type plants would eliminate the
differences with mutant plants.
According to our results, we propose that TCP15 and
related class-I TCP proteins act to attenuate anthocyanin accumulation after short times of exposition to high
light intensity. If high light intensity conditions persist,
inhibition of anthocyanin biosynthesis by TCP15 is
progressively relieved by oxidation of the protein. This
82
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Redox Modulation of TCP15 by High Light Intensity
would prevent excessive anthocyanin accumulation after
short periods of high light intensity but allow a proper
protective response under persistent high illumination. In
addition, since TCP15 and other class-I TCP proteins are
developmental regulators, high light-intensity-dependent
inhibition of class-I TCP protein action may transduce
changes in light intensity into morphological or developmental responses. Finally, the redox-dependent modulation of class-I TCP protein action described here for
anthocyanin biosynthesis under high light intensity conditions may also operate to adjust the action of TCP proteins under other conditions that change the cellular redox
state.
MATERIALS AND METHODS
Plant Materials
Arabidopsis Columbia-0 (Col-0) was used as the wild-type. Plants expressing
TCP15 fused to the EAR repressor domain (Hiratsu et al., 2003) under the control
of the TCP15 promoter (pTCP15:TCP15-EAR) were reported previously
(Uberti-Manassero et al., 2012). The tcp14-4 tcp15-3 double mutant was kindly
provided by Dr. Simona Masiero (Universitá degli Studi di Milano, Italy) and was
described previously (Kieffer et al., 2011). The DR5:GUS reporter was obtained
from the Arabidopsis Biological Resource Center (Ohio State University).
DNA Constructs and Plant Transformation
To generate 35S:TCP15 and 35S:C20S-TCP15 plants, the TCP15 coding sequence or a mutated version encoding Ser at position 20 of the TCP domain
(Viola et al., 2013) were amplified using primers TCP15-F and TCP15-R
(Supplemental Table S2). The amplified PCR products were digested with XbaI
and XhoI and inserted into a modified binary plasmid pBi121 (which contains an
XhoI site at the end of the GUS coding sequence) under the control of the
35SCaMV promoter. To obtain lines expressing TCP15 fused to RFP under the
control of the 35SCaMV promoter, the TCP15 coding sequence was cloned into
entry vector pENTR-3C (Life Technologies) and then transferred to destination
vector pGWB554 (Nakagawa et al., 2007), using the Gateway cloning system
(Life Technologies). This vector allows C-terminal fusions of proteins to monomeric red fluorescent protein (mRFP or mCherry). The primers used are listed in
Supplemental Table S2. All constructs were checked by DNA sequencing and
introduced into Agrobacterium tumefaciens strain LB4404. Arabidopsis plants were
transformed by the floral dip procedure (Clough and Bent, 1998). Transformed
plants were selected on the basis of kanamycin resistance and genotyping.
35S:TCP15 x DR5:GUS plants were obtained by crossing 35S:TCP15 (line 2)
plants with DR5:GUS plants, followed by selfing and selection based on kanamycin
resistance. The presence of DR5:GUS in the genome was confirmed by PCR analysis
of genomic DNA with primers GUS-F and GUS-R (Supplemental Table S2).
Plant-Growth Conditions
Plants were grown in soil at 22 to 24°C under a long-day photoperiod (16 h of
illumination by a mixture of cool-white and GroLux fluorescent lamps) at an
intensity of 100 mE m22 s21. For high light intensity treatments, 2-week-old
Arabidopsis plants were illuminated by light at 800 mE m22 s21, obtained by
supplementing normal light with a metal-halide lamp, under a short-day (8 h of
illumination) photoperiod. The high light intensity treatment was started at the
middle of the illumination period of d 1. Unless otherwise stated, samples were
collected at the end of the light period of each day. Experiments were performed
in two different growth chambers and we observed that levels and rates of anthocyanin accumulation varied between them, probably because of differences in
ambient conditions. The effects described here for the different plant lines under
study, however, were consistently observed under both growth conditions.
Anthocyanin Measurement
Anthocyanin measurement was performed using a method adapted from
Neff and Chory (1998). Seventy-five milligrams of tissue were collected from
three plants in three replicates. Tissue was ground into a fine powder in liquid
nitrogen, extracted with 1 mL of methanol acidified with 1% HCl, incubated
overnight at 4°C, and centrifuged at 12,000 rpm for 5 min. Distilled water
(0.25 mL) was added to 0.3 mL of supernatants and anthocyanins were separated
from chlorophylls by extraction with 0.5 mL of chloroform. Total anthocyanin
content in the aqueous phase was measured at 529 nm in a spectrophotometer and
the amount of anthocyanin was calculated as mg/gram of fresh weight using a
molar extinction coefficient of anthocyanin of 30,000 l/(mol 3 cm) and a Mr of 449.2.
Gene-Expression Analysis
Total RNA was extracted from entire rosettes with Trizol reagent (Invitrogen). One microgram of total RNA was used for cDNA synthesis with oligo
(dT) primer and MMLV reverse transcriptase (Promega) under standard conditions. PCR was performed with an aliquot of the cDNA synthesis reaction with
primers specific for the genes under analysis in 20 mL final volume containing
1 mL SYBR Green, 10 pmol of primers, 3 mM MgCl2, and 0.2 mL platinum Taq
DNA polymerase (Invitrogen). The amplification was monitored in real time on
an MJ Research Chromo4 apparatus. Based on primer efficiency, fold expression was calculated after normalization to actin (ACT2 and ACT8; Charrier
et al., 2002) by a comparative Ct method. Triplicate PCR reactions of single lines
were analyzed for each gene. Similar results were obtained with at least two
additional lines in each case. The primers used for PCR are listed in
Supplemental Table S2. GUS staining was performed as described in UbertiMansassero et al. (2012).
Microarray Results
The microarray experiment was performed in triplicate with total RNA
isolated from rosettes of 25-day-old wild-type and p15:TCP15-EAR plants
grown under standard conditions using Agilent Arabidopsis (V4) 4344K arrays
(two samples in two-color arrays with reciprocal labeling plus one sample in
one-color arrays). The data are deposited in the GEO database under Accession
nos. GSE57742, GSE57743, and GSE57744. For the analysis of differentially
expressed genes, intensities were background-corrected employing the “normexp” method with an offset of 16, except for those probes with log2 background intensity above 10, which were excluded. After normalization using the
loess procedure (for two-color arrays) or quantile normalization (for one-color
arrays), a MA object (Minus/Average of log2 intensities) was generated. Differentially expressed genes were identified as those with absolute log2 fold
change above 1 after applying a FDR-adjusted p-value of 0.05. Genes related
with anthocyanin metabolism were extracted manually from the list of differentially expressed genes.
DNA-Binding Assays
For EMSAs, 10 mg of total protein extracts from transgenic plants prepared
as described previously in Viola et al., 2013 were incubated with 50 ng of
dsDNA generated by hybridization of the complementary oligonucleotides
59-AATTCAGATCTGTGGGACCGGGAG-39 (59-end labeled with FAM) and
59-GATCCTCCCGGTCCCACAGATCTG-39 (TCP15 binding site underlined).
Binding reactions were performed in 20 mM HEPES (pH 7.5), 50 mM KCl, 2 mM
MgCl, 0.5 mM EDTA, 0.5% Triton X-100, 1 mg of poly(dI-dC), and 10% glycerol.
After incubation on ice for 20 min, the reactions were supplemented with 2.5%
Ficoll and immediately loaded onto a running gel (5% acrylamide, 0.08% bisacrylamide in 0.53 TBE plus 2.5% glycerol; 13 TBE is 90 mM Tris-borate, pH 8.3,
2 mM EDTA). The gel was run in 0.53 TBE at 20 mA for 2 h and directly scanned
for fluorescence using a Typhoon scanner (GE Healthcare Life Sciences). For
competitions, complementary oligonucleotides with changes in positions 3
and 8 of the class-I TCP binding site (59-GTGGGACC-39), 59-AATTCAGATCTGTAGGACTGGGAG-39 and 59-GATCCTCCCAGTCCTACAGATCTG-39,
were used.
Western Blot Analysis
For Western blot analysis, 45 mg of protein extracts were separated through
reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then
transferred to Hybond-ECL membranes (GE Healthcare Life Sciences). Membranes were subsequently probed with an antibody against RFP (Living colors
DsRed Polyclonal Antibody, Clontech) at a dilution of 1:1,000, and developed
with anti-rabbit immunoglobin conjugated with horseradish peroxidase using
the SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Plant Physiol. Vol. 170, 2016
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Viola et al.
Accession Numbers
Microarray data from this article can be found in the GEO database under
Accession nos. GSE57742, GSE57743, and GSE57744.
Supplemental Data
The following supplemental materials are available.
Supplemental Table S1. Genes involved in anthocyanin biosynthesis
showing significantly altered expression in p15:TCP15-EAR plants.
Supplemental Table S2. Oligonucleotides used in this study.
Supplemental Figure S1. Anthocyanin levels in extracts from wild-type
and p15:TCP15-EAR plants after a high light intensity treatment.
Supplemental Figure S2. TCP15 transcript levels in 35S:TCP15 and 35S:
C20S-TCP15 plants.
ACKNOWLEDGMENTS
We thank the Arabidopsis Biological Resource Center, Martin Kieffer, and
Simona Masiero for seed stocks. We also thank Elina Welchen and Leandro
Lucero, from our lab, for generating the 35S:TCP15-RFP line and Agustin Arce
for help with the analysis of microarray data.
Received July 7, 2015; accepted November 15, 2015; published November 16,
2015.
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