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ORIGINAL RESEARCH
published: 25 August 2015
doi: 10.3389/fpls.2015.00654
Edited by:
Ute Roessner,
The University of Melbourne, Australia
Reviewed by:
Brian Traw,
University of Pittsburgh, USA
Fumiya Kurosaki,
University of Toyama, Japan
*Correspondence:
Tamara Gigolashvili,
Botanical Institute and Cluster of
Excellence on Plant Sciences,
University of Cologne,
BioCenter, D-50674 Cologne,
Germany
t.gigolashvili@uni-koeln.de
Specialty section:
This article was submitted to
Plant Metabolism
and Chemodiversity,
a section of the journal
Frontiers in Plant Science
Received: 15 June 2015
Accepted: 07 August 2015
Published: 25 August 2015
Citation:
Frerigmann H, Glawischnig E
and Gigolashvili T (2015) The role
of MYB34, MYB51 and MYB122
in the regulation of cam alexin
biosynthesis in Arabidopsis thaliana.
Front. Plant Sci. 6:654.
doi: 10.3389/fpls.2015.00654
The role of MYB34, MYB51 and
MYB122 in the regulation of
camalexin biosynthesis in
Arabidopsis thaliana
Henning Frerigmann1, Erich Glawischnig2and Tamara Gigolashvili1*
1Botanical Institute and Cluster of Excellence on Plant Sciences, University of Cologne, Cologne, Germany, 2Lehrstuhl für
Genetik, Technische Universität München, Freising, Germany
The phytoalexin camalexin and indolic glucosinolates share not only a common
evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory
proteins controlling the shared enzymatic steps are also modulated by the same
R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense
metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates
appear to have a common evolutionary origin and are interconnected on the biosynthetic
level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx),
is also required for the biosynthesis of indolic glucosinolates and is under tight control by
the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin
was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant,
suggesting that these transcription factors are important in camalexin biosynthesis.
Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic
and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant
with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely,
tryptophan could not complement the low camalexin phenotype of this mutant, which
supports a role for the three MYB factors in camalexin biosynthesis upstream of
IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3
and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs
could not activate pCYP71B15::uidA expression in trans-activation assays with cultured
Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors
regulating the generation of IAOx as precursor of camalexin.
Keywords: camalexin biosynthesis, transcriptional regulation, MYB51, MYB122, MYB34
Introduction
Phytoalexins are important defense compounds produced by plants in response to infection by
a large variety of microorganisms. However, the elucidation of camalexin biosynthesis benefited
from the fact that abiotic elicitors like silver nitrate (AgNO3;Glawischnig et al., 2004)andUV
(Müller et al., 2015) strongly induce the camalexin production. Camalexin (3-thiazol-2-yl-indole)
is an indole alkaloid phytoalexin that is specific to a group of cruciferous species including the
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
model plant Arabidopsis thaliana, but is absent in more distantly
related Brassicaceae species (Glawischnig, 2007;Rauhut and
Glawischnig, 2009;Bednarek et al., 2011). The induction of
camalexin biosynthesis genes is strictly localized to sites of
pathogen application, as demonstrated by quantitative RT-PCR
and reporter-gene analysis and there is no evidence existing
for long-distance camalexin transport (Schuhegger et al., 2007).
During camalexin biosynthesis, tryptophan (Trp) is converted to
indole-3-acetaldoxime (IAOx; Figure 1). This step is shared with
the biosynthesis of other Trp-derived metabolites andis catalyzed
by two homologous cytochrome P450 enzymes, CYP79B2, and
CYP79B3 (Hull et al., 2000;Mikkelsen et al., 2000;Zhao et al.,
2002). The resulting IAOx is a precursor of camalexin, indolic
glucosinolates (IGs) and indole-carboxylic acids (ICAs; Böttcher
FIGURE 1 | Regulation by MYB transcription factors in the camalexin biosynthesis pathway. Proven positive transcriptional regulation is shown by dotted
lines with arrows. Modified from Böttcher et al. (2009), Geu-Flores et al. (2011), and Müller et al. (2015); indolic glucosinolates (IG); glutathione (GSH);
dihydrocamalexic acid (DHCA).
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
et al., 2014). In camalexin biosynthesis, IAOx is dehydrated
to indole-3-acetonitrile (IAN) by CYP71A12 and CYP71A13
(Figure 1;Müller et al., 2015). In accordance with their specific
function in phytoalexin biosynthesis, both corresponding genes
are expressed at very low levels in the absence of stress and
are induced by pathogen infection, application of pathogen-
associated molecular patterns (PAMPs), or by AgNO3(Nafisi
et al., 2007;Millet et al., 2010). IAN is also generated during
the degradation of glucobrassicin (I3M; Burow et al., 2008)
and it can be converted to indole-3-carbaldehyde (ICHO) and
ICA by CYP71B6 (Böttcher et al., 2014)(Figure 1). Under
specific conditions IAN serves also as a precursor for IAA
(Kutz et al., 2002;Park et al., 2003). However, the IAN pools
seem to be strictly seperated, thus IAN from I3M breakdown
cannot serve as a precursor of camalexin, but only for ICAs,
as it was shown with a TALEN generated cyp71A12 cyp71a13
double knockout (Müller et al., 2015). Notably, the cyp79b2/b3
double knockout mutant cannot synthesize camalexin (Zhao
et al., 2002;Glawischnig et al., 2004), but this ability was recovered
in a chemical complementation strategy by feeding the mutant
with camalexin precursors such as IAN and dihydrocamalexic
acid (DHCA; Schuhegger et al., 2006;Nafisi et al., 2007;Böttcher
et al., 2009). In camalexin biosynthesis IAN is conjugated with
glutathione (Nafisi et al., 2007;Parisy et al., 2007;Böttcher et al.,
2009;Su et al., 2011). From this glutathione conjugate (GS-IAN)
a cysteine conjugate Cys(IAN) is formed, involving γ-Glutamyl
Peptidases 1 and 3 (GGP1/3; Geu-Flores et al., 2011), which is
the substrate for CYP71B15/PAD3 (Zhou et al., 1999;Schuhegger
et al., 2006;Böttcher et al., 2009).
Although the pathway leading to camalexin has been
largely elucidated, its regulation remains less well understood.
Perception of fungal pathogens such as Botrytis cinerea
(Kliebenstein et al., 2005)andAlternaria alternata (Schuhegger
et al., 2007) significantly activates camalexin production via
mitogen-activated protein kinase (MAPK) cascade, which in
turn phosphorylates MPK3 and MPK6 (Ren et al., 2008).
Camalexin synthesis is almost completely blocked in the mpk3/6
double mutant after infection by B. cinerea (Ren et al., 2008).
Mao et al. (2011) have demonstrated that the Arabidopsis
transcription factor WRKY33 is a molecular target of the
MPK3/6 cascade. wrky33 mutant can synthesize only very low
amounts of camalexin, even in the MPK3/6 gain-of-function
mutant. Furthermore, MPK4 physically interacts with MPK4
SUBSTRATE 1 (MKS1) and WRKY33 and represses WRKY33
function. Activated MPK4 phosphorylates MKS1, which in
turn, releases WRKY33, which can then bind to the promoter
of CYP71B15 (Qiu et al., 2008). Surprisingly, the respective
wrky33 knock-out mutant contains low camalexin levels only
at early stages of infection, but at later stages, contains even
more camalexin than wild-type (WT) plants (Birkenbihl et al.,
2012). Together, these results indicate that WRKY33 is one
important regulator of camalexin, but that other regulators
exist.
The transcription of NAC042 is strongly induced by AgNO3,
a known inducer of camalexin biosynthesis, and the nac042
null mutant accumulates about 50% of WT camalexin levels
after treatment with AgNO3or B. cinerea (Saga et al.,
2012). Furthermore, the induction of camalexin biosynthesis
by acifluorfen, which generates reactive oxygen species (ROS),
results in about 15% of the WT camalexin level in nac042,
which highlights the key role of NAC042 in the ROS-
dependent induction of camalexin biosynthesis (Saga et al.,
2012).
To synthesize camalexin, it is essential that the specific
genes (CYP71A12,CYP71A13,andCYP71B15) are upregulated
together with the upstream Trp biosynthetic genes and CYP79B2.
The known regulator of camalexin, WRKY33, binds to the
promoters of CYP71B15 and CYP71A13 (Birkenbihl et al.,
2012), whereas the regulators of IG biosynthesis, MYB34,
MYB51, and MYB122 control genes of the shikimate pathway
to Trp, i.e., anthranilate synthase αand βsubunits, Trp
synthases and CYP79B2 (Gigolashvili et al., 2007;Malitsky
et al., 2008;Frerigmann and Gigolashvili, 2014). These MYB
transcription factors thus positively regulate all the necessary
steps for the production of the camalexin precursor IAOx. In
addition to this intermediate, IG and camalexin biosynthesis
share a glutathione conjugation step and the involvement
of GGP1 (Geu-Flores et al., 2011), reflecting that camalexin
biosynthesis likely has evolved from IG biosynthesis (Rauhut
and Glawischnig, 2009;Bednarek et al., 2011). Therefore,
MYB34, MYB51, and MYB122 possibly not only regulate
the IG biosynthesis pathway, but also activate genes in
the closely related camalexin biosynthesis pathway. Here we
addressed the potential involvement of three MYB transcription
factors in camalexin biosynthesis and show that especially
MYB51 and MYB122 are involved in camalexin biosynthesis,
because its synthesis is strongly reduced in corresponding
double and triple mutants. Metabolite complementation of
the triple myb34/51/122 mutant reveals the importance of
these MYBs in the regulation of camalexin biosynthesis
upstream of IAOx. Thus, camalexin and IGs not only possess
a tightly interconnected biosynthetic pathway, but are at
least partially regulated by the same R2R3-MYB transcription
factors.
Results
Camalexin Biosynthesis Genes are
Co-expressed with MYB51 and MYB122
Camalexin biosynthesis is induced locally by exposure to biotic
or abiotic stresses and the genes involved in its biosynthesis are
highly co-ordinately expressed. To address the role of MYB34,
MYB51, and MYB122 in camalexin biosynthesis, we exploited
existing co-expression databases like ATTED1(Obayashi et al.,
2009). The survey revealed that MYB51 and MYB122 are not
only co-regulated with genes for Trp and IAOx biosynthesis,
but also with CYP71B15/PAD3,CYP71A12,andCYP71A13
(Supplementary Tables S1 and S2). This implicates both MYB
factors as good candidate regulators of camalexin biosynthesis in
Arabidopsis.
1http://atted.jp/
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
MYB51 and MYB122 are Induced by Silver
Nitrate and by Pathogen-Associated Molecular
Pattern (PAMP) from Pythium
aphanidermatum (PaNie)
To further validate the importance of R2R3-MYBs in camalexin
regulation, we analyzed the induction of MYB34,MYB51,and
MYB122 in response to elicitors of camalexin production. In a
pilot experiment, we treated Arabidopsis Col-0 WT plants with
AgNO3, a commonly used abiotic elicitor of camalexin induction,
which strongly induced CYP71B15, CYP71A13,MYB122,and
MYB51 (Figure 2). However, the expression of MYB34 was
reduced, indicating that it plays a less important role in
phytoalexin regulation.
In addition, we analyzed transgenic plants that expressed
a gene encoding a Nep1-like protein from Pythium
aphanidermatum (PaNie), which acts as a PAMP, under the
control of an ethanol-inducible promoter (Rauhut et al., 2009).
The production of this Nep1-like protein triggers the strong
accumulation of camalexin 8 h following ethanol inductions
(Rauhut et al., 2009). The transcription of MYB51 significantly
FIGURE 2 | Silver nitrate induces the transcription of MYB51 and
MYB122 as well as that of CYP71B15 and CYP71A13. The expression of
camalexin biosynthesis genes (CYP71B15 and CYP71A13)andofMYB34,
MYB51,andMYB122 upon silver nitrate (AgNO3) treatment is shown. The
relative expression in Col-0 was measured in leaves of 6-week-old plants 18 h
after treatment (MOCK =1). Data are means ±SE from four independent
experiments each with two to three biological replicates (n=11). Values
marked with asterisks are significantly different from those of control plants
(Student’s t-test; p<0.05).
increased 150 min after treatment, and that of CYP71B15,
CYP71A13,andMYB122 after 300 min (5 h; Figure 3).
Conversely, MYB34 was not induced by PaNie expression,
which confirms its minor role in camalexin regulation. A similar
induction pattern of the MYBs and camalexin genes was observed
upon colonization with the fungus Piriformospora indica
(Lahrmann et al., 2015). In addition, MYB51 transcription was
also increased 40 and 88 h after infection with the necrotrophic
pathogen B. cinerea,asrevealedbythepMYB51::GUS reporter
(Supplementary Figure S1).
Taken together, the expression patterns of MYB51 and
MYB122 implicate a role in camalexin biosynthesis.
The Induction of MYB51 and MYB122 upon
Wounding Coincides with that of the
Camalexin Biosynthesis Gene CYP71B15
Wounding of the plant surface creates a potential entry point for
invading pathogens, and plant response to injury by localized
defense responses includes the induction of defense-related
genes and the accumulation of anti-microbial proteins such as
proteinase inhibitors, chitinase, or glucosinolates (Ryan, 1990;
Chang et al., 1995;Reymond et al., 2000;Chassot et al., 2008).
Especially strong wounding releases oligogalacturonides from the
plant cell wall which can induce a local defense response similar
to bacterial PAMPs (Denoux et al., 2008). Thus wounding of
Arabidopsis leaves hasbeen previously shown to lead to immunity
to B. cinerea, because hyphal growth on wounded plants was
significantly inhibited in comparison to that on unwounded
controls (Chassot et al., 2008).
To address the involvement of the MYB34, MYB51, and
MYB122 transcription factors in wounding response, the
transcription of their respective genes was analyzed 1, 5, 10,
30, 120, and 300 min after strong wounding. Wounding of WT
Arabidopsis leaves increased the transcription of MYB51 and
MYB122, but not of MYB34 after 120 min of injury (Figure 4),
which represented the time-point of increased expression of the
camalexin biosynthesis gene CYP71B15 (Figure 3). This second
induction peak of MYB51 and MYB122 transcription appears to
be related to induction of camalexin biosynthesis. During the
second phase of the wounding response, the transcript levels
of MYB34 decreased, whereas expression of MYB51,MYB122,
and CYP71B15 continued to increase and remained high even
at 300 min (5 h) of treatment (Figure 4). The first peak in MYB
transcription recorded after 5–10 min of injury observed in this
study (Figure 3)andpreviously(Gigolashvili et al., 2007), was
associated with an increase in IG biosynthesis.
In order to confirm that the applied strong wounding does
not resemble solely jasmonate signaling, as it is known for
standard wounding application, hormone marker genes for
jasmonate (VSP2), salicylic acid (PR1), and ethylene/jasmonate
(PDF1.2) were checked (Supplementary Figure S2). As expected
no induction, but even a repression of VSP2 was observed,
while PR1 and PDF1.2 transcript levels increased similar to
oligogalacturonide treatment (Denoux et al., 2008).
Together, these data suggest a role for MYB51 and MYB122
in priming camalexin biosynthesis at later stages of wounding
response, to protect against biotic and abiotic stressors.
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
FIGURE 3 | Induction of MYB51 and MYB122 in rosette leaves of
Alc::PaNieDc plants. Expression of MYB34 (A),MYB51 (B),MYB122 (C),
CYP71A13 (D), and the camalexin biosynthesis gene CYP71B15 (E) following
AgNO3treatment. Relative expression in pAlc::PaNieDc was measured in leaves
of 6-week-old plants induced with ethanol (for 60 min, 150 min or 300 min; time
point 0 =1 min). Data are means ±SE from two independent experiments
each with three biological replicates (n=6). Values marked with asterisks are
significantly different from those of control plants (Student’s t-test; p<0.05).
FIGURE 4 | Wounding response of MYB34,MYB51, and MYB122 in
leaves. Detached leaves of 6-week-old Col-0 plants grown under short day
conditions were strongly wounded. Leaves were harvested after 1, 5, 10, 30,
120, and 300 min and processed for transcript analysis by qPCR. Relative
transcript levels for MYB34, MYB51,MYB122,andCYP71B15 are shown for
wounded vs. unwounded leaves (time-point 0 =1 min). Data are means ±SE
from three independent cultivations each with two biological replicates (n=6).
Values marked with asterisks are significantly different from the 0 time point
(Student’s t-test; p<0.05).
myb Mutants are Impaired in UV-Dependent
Camalexin Induction
The abiotic elicitor UV can be easily applied to uniformly
trigger camalexin induction in Col-0 (Müller et al., 2015 and
Supplementary Figure S3B). We tested double and triple loss-
of-function mutants of MYB51,MYB122,andMYB34 for
their ability to synthesize camalexin after UV treatment. The
camalexin content of leaves of double myb51/122,myb34/51,
and triple myb34/51/122 mutants was strongly reduced after
18 h UV treatment (Figure 5), suggesting an important
function for all three MYBs and especially MYB51 in camalexin
accumulation. The myb34/122 double mutant showed only a
minor and statistically non-significant reduction in camalexin
accumulation. Camalexin levels were significantly lower in the
triple myb34/51/122 mutant than in WT plants, but not in
comparison to that of the myb34/51 and myb51/122 mutants.
However, 24 h after UV treatment, only the myb51/122 double
mutant and myb34/51/122 triple mutant contained significantly
less camalexin than the WT (Supplementary Figure S3). These
camalexin levels in myb mutant backgrounds confirm the
importance of MYB51 and MYB122 in camalexin accumulation.
The role of MYB34 appears to be minor.
The Camalexin Biosynthesis Genes CYP71B15
and CYP71A13 are not Downregulated in the
Triple myb34/51/122 Mutant
The myb34/51/122 triple mutant is limited in the synthesis of
IAOx, a precursor of IGs and camalexin and consequently,
IGs (Frerigmann and Gigolashvili, 2014)andcamalexinare
reduced (Figure 5A). To investigate the role of MYBs on the
expression of camalexin biosynthesis genes, the steady-state
mRNA levels of CYP71B15 and CYP71A13 were analyzed in the
triple myb34/51/122 mutant. If MYB51, MYB122, and MYB34
directly regulate camalexin biosynthesis genes, the expression of
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
FIGURE 5 | The UV-dependent induction of camalexin is impaired in
multiple myb knock-out mutants. (A) The relative amount of camalexin after
18 h UV treatment in Col-0 and double and triple myb mutants (Col-0 =100%).
Data are means ±SE from two independent cultivations each with six biological
replicates (n=12). (B) The relative amount of camalexin 18 h after UV treatment
in myb34/51/122 fed with H2O, or 0.25 mM Trp, IAOx or IAN (Col-0 =100%).
Data are means ±SE from three independent cultivations each with six
biological replicates (n=18). Different letters indicate significant differences at
p<0.05 (Kruskal Wallis Test, followed by a Mann Whitney UTe s t wi th
Bonferroni-corrected p-values; p<0.05).
these genes should be significantly decreased in myb34/51/122,
similar to that of IG biosynthesis genes.
Genes involved in IAOx biosynthesis were strongly down
regulated in the myb34/51/122 mutant (Figure 6), whereas
the expression of CYP71B15 and CYP71A13 either remained
unchanged or increased. This increase in specific camalexin gene
expression is not accompanied by higher levels of camalexin
in the mutants (Supplementary Figure S3). The activity of
pCYP79B2:uidA increased, whereas that of pCYP71B15:uidA
was not affected by all three MYB factors, as demonstrated
by co-expression via trans-activation assays with cultured
Arabidopsis cells (Berger et al., 2007)(Figure 6B). Conversely,
WRKY33, the transcription regulator of CYP71B15,induced
pCYP71B15::uidA when co-expressed with p35S:WRKY33
in cultured cells. Thus, MYBs do not directly regulate these
important camalexin biosynthesis genes downstream of
IAOx.
We also attempted to metabolically complement the low-
camalexin phenotype of myb34/51/122 mutant leaves upon
UV-treatment,byfeedingthemwithIAOx,IANorTrp.
Treatment with IAOx or IAN partially restored camalexin levels
in the myb34/51/122 mutant upon UV-treatment, whereas Trp
feeding did not (Figure 5B). Because Trp could not complement
the low-camalexin phenotype of the triple myb mutant, we
conclude that the three MYB factors studied essentially regulate
the synthesis of IAOx from Trp, but are not directly involved in
the activation of genes downstream of IAOx.
Discussion
The camalexin biosynthetic pathway has been largely elucidated,
but little is known about the regulatory components of this
pathway. WRKY33 binds to the promoters of CYP71B15 and
CYP71A13 to activate camalexin biosynthesis, but also other
regulators have to be involved, because its loss of function leads
to low camalexin levels only during early stages of pathogen
infection (Birkenbihl et al., 2012). In this study, we addressed
the role of the known IG regulators MYB34, MYB51, and
MYB122 in the biosynthesis of camalexin in Arabidopsis.Because
the camalexin and IG biosynthetic pathways have a common
evolutionary origin and are tightly interconnected, these two
classesofcompoundsmightberegulatedbythesamesetof
transcription factors.
MYB51 and MYB122 are Induced by Biotic and
Abiotic Triggers of Camalexin Biosynthesis
Camalexin biosynthesis is induced in plants following exposure
to abiotic stresses such as heavy metal treatment or UV-C
radiation or exposure to pathogens. We addressed the role
of MYB34, MYB51 and MYB122 in camalexin biosynthesis
by analysing their mRNA levels in plants exposed to several
camalexin-inducing agents. Treatment of Arabidopsis WT plants
with the abiotic elicitor AgNO3caused a significant increase
in steady-state mRNA levels of MYB122 and MYB51,butnot
of MYB34. Similarly, MYB122 and MYB51 were induced in
transgenic plants that expressed a NEP1-like protein from PaNie
under the control of an ethanol-inducible promoter (Rauhut
et al., 2009), endorsing the possible role of these two transcription
factors in camalexin biosynthesis. In addition, the MYB51
promoter was also induced after treatment with B. cinerea
(Supplementary Figure S1). Finally, wounding of leaves, which
is known to provide a protection against B. cinerea by priming
camalexin production in Arabidopsis (Chassot et al., 2008),
increased the expression of MYB51 and MYB122. Two induction
peaks of MYB expression in response to wounding within the
analyzed time scale occurred: the first peak in wounding response
of MYB51 transcript level was observed after 5–10 min of injury
(Gigolashvili et al., 2007) and was associated with a switch
in the IG biosynthesis machinery, and the second phase of
induction concerned the transcription of MYB51 and MYB122,
but not MYB34, after 120 min of injury, which coincided with
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Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
FIGURE 6 | The presence of solely MYB34,MYB51,orMYB122 is not
enough to activate the pCYP71B15:uidA and pCYP71A13:uidA in trans.
(A) The expression of specific genes for camalexin biosynthesis (CYP71B15 and
CYP71A13) and genes underlying the conversion of tryptophan to IAOx
(CYP79B2 and CYP79B3) was analyzed in the myb34/51/122 mutant. Relative
expression was measured in leaves of 6-week-old plants (Col-0 =1). Data are
means ±SE from three independent cultivations with three biological replicates
(n=9). Values marked with asterisks are significantly different from those of
control plants (Student’s t-test; ∗p<0.05). (B,C) Tra n s -activation with MYB34,
MYB51,andMYB122 and target promoters of the camalexin biosynthesis
pathway genes CYP79B2, CYP71A13,andCYP71B15.(B) The
promoter–reporter constructs of pCYP71A13:uidA,pCYP71B15:uidA or
pCYP79B2:uidA were co-expressed in the same cells with effector constructs
p35S:MYB34,p35S:MYB51,orp35S:MYB122. The cultured A. thaliana cells
were inoculated with the supervirulent Agrobacterium tumefaciens strain
LBA4404.pBBR1MCS.virGN54D, containing either only the reporter construct
or the reporter and effector construct in a 1:1 ratio. The GUS staining indicates
trans-activation of the promoter by the effector. (C) The trans-activation
potential of the p35S:WRKY33 effector toward the promoters of CYP71A13 and
CYP71B15.
an strongly increased expression of the camalexin biosynthesis
gene CYP71B15 (Figure 3). This second phase might therefore be
related to camalexin biosynthesis. According to directed studies
(Schuhegger et al., 2006;Chassot et al., 2008) and to the analysis
of microarray data (see, e.g., efp browser analysis of ATH1
Affymetrix data2;Winter et al., 2007)CYP71B15 did not show
strong responsiveness to wounding. In this light it was surprising
that here CYP71B15 expression was induced more than 100-fold.
We here applied rather harsh and extensive wounding to the
tissue. Possibly severe wounding induces camalexin biosynthesis
by eliciting oligogalacturonides, which originate from the plant
cell wall (Denoux et al., 2008), while restricted wounding has a
minor effect.
Taken together, the induced expression of MYB51
and/or MYB122 after exposure to biotic and abiotic
triggers of camalexin biosynthesis [AgNO3,wounding,
PAMP (PaNie) and the necrotrophic pathogen B. cinerea]
suggests that the transcription factors encoded by these
genes play a role in camalexin biosynthesis. Because
the expression of MYB34 was not affected by the same
treatments, we conclude that it is not involved in camalexin
biosynthesis.
2http://bar.utoronto.ca/efparabidopsis/cgi-bin/efpWeb.cgi
The Role of MYBs in the Regulation of IAOx – A
Branch-Point in IG, Camalexin, ICA, and IAA
Synthesis
The initial step of camalexin, IG, and ICA biosynthesis is
the conversion of Trp to IAOx mediated by CYP79B2 and
CYP79B3. The interplay between IAOx-derived metabolites was
also demonstrated by the analysis of mutants deficient in IG
biosynthesis. The loss of function of IG biosynthetic genes
downstream of IAOx (cyp83b1/sur2,C-S lyase/sur1,andugt74b1
null mutants) results in a strong auxin-overproducing phenotype
(Barlier et al., 2000;Grubb et al., 2004;Mikkelsen et al., 2004).
This is possibly due to IAOx accumulation in cells because of
“biosynthetic blockage” in the IG pathway, and consequently,
unspecific conversion of excess IAOx to the auxin IAA. Other
metabolites such as ICA and camalexin, which can be also
induced in these mutants, were not analyzed in the above-
mentioned studies.
In the WT, biosynthesis of IAOx is under tight transcriptional
control by MYB34, MYB51 and MYB122 transcription factors
(Celenza et al., 2005;Gigolashvili et al., 2007;Frerigmann
and Gigolashvili, 2014). Consequently the MYBs, especially
the MYB51 and MYB122 are considered as candidates in the
regulation of other Trp-derived metabolites than IGs, e.g.,
camalexin. We propose the following scenario for the role
Frontiers in Plant Science | www.frontiersin.org 7August 2015 | Volume 6 | Article 654
Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
of MYB34, MYB51, and MYB122 in camalexin biosynthesis:
they regulate genes involved in camalexin biosynthesis similar
to how they regulate IG production. However, they have
to act in concert with other regulators, since they are also
highly expressed and regulate IG production in non-triggered
tissue and would therefore lead to camalexin accumulation
in healthy plants. We therefore suggest that specific signaling
components exist upstream to these MYB factors, including
alternative transcription factors, which activate different sets
of genes for camalexin and IG biosynthesis. These different
signaling components are responsive to AgNO3,PAMPs,
and UV in the case of camalexin biosynthesis, and to
herbivores regarding IG production. Thus, to enable camalexin
biosynthesis, the MYBs and additional transcription factors
are activated: MYB factors regulate IAOx biosynthesis, and
alternative (unknown) regulators, together with WRKY33,
control camalexin genes downstream of IAOX (e.g., CYP71B15
and CYP71A13).
The Regulation of Camalexin Biosynthesis by
MYB51, MYB122, and MYB34
The analysis of camalexin accumulation in higher-order loss-
of-function mutants of MYB51, MYB122,andMYB34 treated
with UV revealed a strong reduction in the camalexin content of
leaves of double myb51/122, myb34/51,andtriplemyb34/51/122
mutants (Figure 5), emphasizing the importance of MYB51
in camalexin accumulation in Arabidopsis. The role of MYB34
for camalexin induction was negligible, whereas MYB122
contributes camalexin biosynthesis, as demonstrated by the
response of higher-order myb mutants after 24 h treatment
with UV (Supplementary Figure S3). We propose the following
explanation for the observed role of MYB122:(i)MYB122 is
the lowest-expressed gene among the three MYBs (Frerigmann
and Gigolashvili, 2014), therefore, the observed metabolic
effects reflect its transcript abundance; (ii) transcription of
MYB122 is positively regulated by MYB51, which is essential
for camalexin biosynthesis. This positive correlation between
MYB51 and MYB122 expression due to reciprocal regulation
was previously demonstrated by the analysis of myb knock-
out and overexpression plants (Frerigmann and Gigolashvili,
2014). The reciprocal activation of mRNAs of these two MYBs
might play an important role in the regulation of camalexin
biosynthesis.
To elucidate further the role of the MYBs, we performed
a metabolic complementation experiment by feeding the UV-
treated leaves of the camalexin-deficient mutant myb34/51/122
with the precursors Trp, IAOx, or IAN (Figure 5B). This
experiment demonstrated that the three MYBs are essential
to regulate the synthesis of IAOx from Trp during camalexin
biosynthesis. However, they are not directly involved in the
activation of genes downstream of IAOx, because both IAOx
and IAN could complement the low camalexin phenotype
of the myb34/51/122 mutant. These experiments suggest the
possibility that MYB51 and MYB122 are indirectly involved in
the activation of CYP71B15 or CYP71A13 by forming dynamic
regulatory complexes with other transcription factors. However,
even if the MYB factors interact with other transcription factors
that regulate camalexin biosynthesis, they are not thought to
activate CYP71B15 or CYP71A13. In support of this, qRT-PCR
analysis of the triple myb34/51/122 mutant and the promoter–
effector assays in cultured cells suggested that CYP71B15 and
CYP71A13 are regulated independently from the MYB genes
(Figure 6).
Taken together, the data substantiate the importance of
three MYB factors in the regulation of camalexin biosynthesis
by providing the precursor metabolite IAOx (Figure 1).
There is no evidence for the direct MYB-mediated regulation
of camalexin biosynthesis genes downstream of IAOx. The
identification of the possible role of MYB51 and MYB122 in the
activation of CYP71B15 or CYP71A13 in complex with other,
yet to be identified transcription factors, is anticipated in the
future.
Experimental Procedures
Arabidopsis Lines Used in this Study
The Arabidopsis loss-of-function mutants used in this study
are all in the Columbia-0 (Col-0) genetic background. The
T-DNA insertion mutants for MYB34,MYB51,MYB122
have been previously described and are myb34 [At5g60890;
WiscDsLox424F3; (Frerigmann and Gigolashvili, 2014),
myb51/hig1 (At1g18570; GK228B12; Gigolashvili et al., 2007),
and myb122-2 (At1g74080; WiscDsLoxHs206_04H; Frerigmann
et al., 2014). The multiple mutants were generated and described
by Frerigmann and Gigolashvili (2014).
The ethanol-inducible overexpression line Alc::PaNieDc
(Rauhut et al., 2009)andthepMYB51::GUS reporter line
(Gigolashvili et al., 2007) were generated as described.
Biotic and Abiotic Treatments of Arabidopsis
Leaves
For treatment with AgNO3, plants were grown for six weeks
under short-day conditions. Pots with five plants were sprayed
with AgNO3or MOCK and harvested after 18 h in the dark
[AgNO3(5 mM AgNO3+0.02% Silver); MOCK (0.02% Silver)].
Expression of the NEP1-like protein in Alc::PaNieDc plants
was induced by spraying with ethanol (2%) or with water for the
MOCK samples. Samples were harvested at four different time
points (0, 60, 150, and 300 min).
For wounding experiments, detached leaves of 6-week-old
Col-0 plants were heavily crushed with forceps on the whole
leaf, additionally strongly wounded with a scalpel and stored
in a petri dish with wet paper tissue till harvest. After 0, 1, 5,
10, 30, 120, and 300 min leaves were frozen in liquid nitrogen
and subsequent directed for RNA isolation and gene expression
analysis by qRT-PCR. Wounding and storage for different time
points had no effect on ACTIN2 levels.
Five-week-old plants were infected with a 6 μL droplet
of B. cinerea spores (2 ×106spores/μLinLB-media)or
LB-media as MOCK. After infection, plants remained under
short-day conditions but with a relative humidity of about 100%.
Samples were harvested at different time points (0, 40, 88 h)
Frontiers in Plant Science | www.frontiersin.org 8August 2015 | Volume 6 | Article 654
Frerigmann et al. Regulation of camalexin biosynthesis by MYBs
and fixed immediately with ice-cold acetone. GUS staining was
performed overnight at 37◦C. Histochemical localisation of GUS
in transgenic plants harboring the pMYB51::uidA construct was
performed as described Gigolashvili et al. (2007).
UV-Treatment, Metabolite Feeding and
Camalexin Measurement
For UV induction, leaves were cut at the base of the petioles
and placed on wet tissue paper under a UV-lamp (Desaga UV-
VIS, λ=254 nm, 8 W) at a distance of 20 cm and were
irradiated for 2 h (Mucha et al., 2015). Camalexin extraction and
HPLC-analysis was performed essentially as previously described
(Schuhegger et al., 2006). Leaves were extracted twice in 200 μl
MeOH/H2O (4:1; v/v) at 65◦C for 30 min. Combined extracts
were centrifuged at 17,000 gfor 15 min and analyzed by
reverse phase HPLC (LiChroCART 250-4, RP-18, 5 μm, Merck;
1mL·min−1; MeOH/H2O (1:1; v/v) for 2 min, followed by a
10 min linear gradient to 100% MeOH, followed by 3 min 100%
MeOH). Camalexin was quantified using a Shimadzu F-10AXL
fluorescence detector (318 nm excitation, 370 nm emission)
and by UV absorption at 318 nm applying a calibration curve
with authentic standard. For intermediate feeding leaves were
detached at the petiole after 2 h UV treatment and incubated in
400 μl 0.25 mM precursor solution or water for an additional
16 h.
RNA Extraction and qRT-PCR
Total RNA extraction and qRT-PCR analysis were as described by
Frerigmann and Gigolashvili (2014).Therelativequantification
of expression levels was performed using the comparative delta
Ct method, and the calculated relative expression values were
normalized to that of ACTIN2 and compared with the expression
level in untreated WT plants (Col-0 =1). When not specified in
the figure legend, three technical replicates and three biological
replicates from independently grown plants were analyzed (for
primer sequences see Supplementary Table S3).
Plant Growth Conditions
Seeds of A. thaliana ecotype Col-0 and mutant lines were
stratified for 2–7 days in the dark at 4◦C to break seed dormancy.
Plants were grown in growth cabinets with a light/dark cycle
of 8 h/16 h and a day/night temperature of 21◦C/18◦C, 40%
humidity and a mean photon flux density of 150 μmol m−2s−1.
A minimum of 100 mg rosette material was harvested from
6-week-old plants, immediately frozen in liquid nitrogen and
kept at –80◦C until RNA extraction or metabolite analysis.
Reporter Construction for Transient
Co-transformation Experiments
The promoter regions of CYP71B15 (At3g26830; from –1,593
to +58 bp) and CYP71A13 (At1g73500; from –2,124 to
+42 bp) were amplified from genomic DNA of Arabidopsis
plants and cloned into the pEntry TOPO vector (Invitrogen).
The construction of the CYP79B2 promoter was performed
as described (Gigolashvili et al., 2007). The corresponding
primer sequences are listed in Supplementary Table S4. The
binary plant transformation vector pGWB3i containing an
intron within the uidA gene was used to drive Agrobacterium-
mediated expression of uidA from these promoters and pGWB3i
was recombined with the pEntry Topo vectors containing
the promoter of interest using LR reactions (Invitrogen). The
final pCYP71B15::uidA,pCYP71A13::uidA and pCYP79B2::uidA
clones in pGWB3i,aswellasp35S::MYB34,p35S::MYB51,
p35S::MYB122,andp35S:WRKY33 in pGWB2 were used to
transform the supervirulent Agrobacterium tumefaciens strain
LBA4404.pBBR1MCS.virGN54D as described by Berger et al.
(2007).
Acknowledgments
We thank Alexandra Chapman and Ulrike Hebbeker for practical
assistance and Dr. John Chandler for critically reading the
manuscript. We also cordially thank Prof. Dr. Ulf-Ingo Flügge
for his continuous support over many years. This work was
financially supported by the Deutsche Forschungsgemeinschaft
(Project Reference Numbers: GI 824/1-1, EXC 1028, and
GL346/5-1, Heisenberg fellowship to EG).
Supplementary Material
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00654
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
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Copyright © 2015 Frerigmann, Glawischnig and Gigolashvili. This is an open-access
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