Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize
et al. (2012) Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for
Arabidopsis and Maize. PLoS ONE 7(5): e36625. doi:10.1371/journal.pone.0036625
Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize
Thomas Hartwig 0
Claudia Corvalan 0
Norman B. Best 0
Joshua S. Budka 0
Jia-Ying Zhu 0
Sunghwa Choe 0
Burkhard Schulz 0
Markus Grebe, Umea Plant Science Centre, Sweden
0 1 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America, 2 School of Biological Sciences, College of Natural Sciences, Seoul National University , Seoul , Korea , 3 Department of Plant Biology, Carnegie Institution for Science, Stanford, California, United States of America, 4 Plant Genomics and Breeding Institute, Seoul National University , Seoul , Korea
Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA3 co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.
Funding: This research was supported in part by grants from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ008051) and the
Cooperative Research Program for Agriculture Science &Technology Development (project no. PJ906910), Rural Development Administration, Republic of Korea,
by Technology Development Program (110033-5) for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (to SC), and
the BK21 Research Fellowships funded by the Ministry of Education, Science, and Technology of the Korean Government (to CC). This work was also supported by
startup funds from the Department of Horticulture and Landscape Architecture, Purdue University (to BS). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Brassinosteroids (BRs) are poly-hydroxylated steroidal
hormones with profound effects on several physiological plant
responses. They are involved in regulating cell elongation and
division , vascular differentiation , photomorphogenesis
, leaf angle inclination , seed germination ,
stomata development , as well as suppression of leaf
senescence and abscission . Radioactive tracer studies in cell
cultures of Catharanthus roseus established the steps of the BR
metabolic pathway . This work was complemented by the
characterization of several BR-deficient mutants in Arabidopsis [14
20], as well as crops like tomato, pea, and rice . These studies
showed that several steps of BR biosynthesis are mediated by
cytochrome P450 monooxygenases (P450s) [13,21]. Although the
importance of BRs for agricultural crops such as sorghum (Sorghum
bicolor) and maize (Zea mays L.) has been recognized , only a
few null-mutations have been reported in these species .
The field of chemical genomics greatly benefited from the use of
chemical inhibitors/modifiers . Potent and specific
biosynthesis inhibitors are useful tools to evaluate the functions of
endogenous substances, including phytohormones. Biosynthetic
mutants and specific metabolic inhibitors displayed their
effectiveness in mode of action studies of gibberellic acid (GA) and BRs [27
Numerous triazole compounds have been shown to inhibit
P450s, one of the largest and most ubiquitous group of plant
enzymes that catalyze oxidative processes in life systems .
Paclobutrazol (Pac) and uniconazole (Ucz) are two triazole plant
growth regulators (Fig. 1) that block sterol 14R-demethylation,
phenocopy GA mutants, and reduce endogenous GA levels .
Both compounds inhibit P450 CYP701, which catalyzes an early
step in GA biosynthesis . Furthermore, Ucz also has been
reported to slightly decrease the endogenous concentration of
castasterone and inhibits BR-induced tracheary element
differentiation . These reports suggested that Ucz may also affects
BR biosynthesis and later screens of structurally similar azoles led
to the development of brassinazole (Brz) (Fig. 1), the first specific
BR biosynthetic inhibitor .
Brzs mode of action is to bind and block DWF4/CYP90B1 .
DWF4 is a P450 that mediates multiple rate-limiting C22
alphahydroxylations in the biosynthesis of BRs . DWF4 expression is a
target of regulation by both endogenous signals like auxin 
and exogenous cues like temperature . Brz and its even more
specific derivative Brz2001  became invaluable tools for BR
research. Not only did they help to reveal the role of BRs in various
plant species , they were also essential for the isolation and
characterization of genes that function in BR signaling .
However, low accessibility and high costs associated with Brz/
Brz2001 have limited their use in agricultural crops that often
require large-scale applications. In this context, it would be
beneficial to the research community to have access to potent,
specific, and more cost efficient azole BR inhibitors in plants.
The triazole compound propiconazole (Pcz), 1-[
(Fig. 1) as a potent inhibitor of BR biosynthesis was first reported
after examining its inhibitory effect on hypocotyl elongation of
cress plants (Lepidium sativum) . This inhibitory effect of Pcz was
reversed by co-application with brassinolide. Based on the Pcz
structure additional BR inhibitors, such as
2RS,4RS-1-[2-(4trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H1,2,4-triazole, were identified . On the other hand, Pcz has
been commercially used as fungistat (BannerMaxx, Syngenta)
against a broad range of phytopathogenic fungi. Its fungistatic
mode of action is the same as that of Ucz and Pac; blocking of
lanosterol 14R-demethylase (CYP51A1) . Pcz has also
been studied extensively for its toxicity on plants, animals, humans,
and the environment . Here we present a molecular
genetic analysis of Pczs effects on Arabidopsis and maize seedlings.
Our results indicate that Pcz is a potent and specific inhibitor of
the BR metabolic pathway in plants.
Arabidopsis seedlings treated with Pcz display dwarf
To study Pczs impact on Arabidopsis, we treated wild-type Ws-2
plants with Pcz concentrations ranging from 0.1 to 5 mM for 5 days.
Cotyledons showed a reduction in size and epinatic growth
responses with treatments of Pcz (Fig. 2AB). Subsequently, we
evaluated the effect of Pcz on primary root length. The results
showed a dose-dependent reduction of primary root growth, where
0.1 mM Pcz decreased the elongation by 20% and 5 mM Pcz by 54%
compared to mock conditions (Fig. 2A, 2C). No further significant
decrease was observed at concentrations higher than 0.5 mM Pcz
We then evaluated the overall efficacy and specificity of Pcz
compared to equal concentrations of Brz and Ucz. Wild-type Ws-2
plants treated with 1 mM Pcz or Brz for 4 days showed comparable
reductions in root length of 38% and 41%, respectively (Fig. 2D). In
contrast, the impact of 1 mM Ucz on reduction of root length was
significantly less with only a 25% decrease compared to mock
treatments (Fig. 2D). We found a complementation of the
phenotypes induced by 1 mM Pcz or Brz, but not 1 mM Ucz, with
co-applications of 0.1 mM 24epi-brassinolide (BL), a bioactive
epimer of brassinolide (Fig. 2D). Root length of plants co-treated
with 1 mM Pcz plus 0.1 mM BL was not significantly different from
mock treatments (Fig. 2D). On the contrary, the co-application of
1 mM Ucz and 0.1 mM BL showed no significant difference to
treatments with 1 mM Ucz alone (Fig. 2D). These results suggest that
Pcz and Brz have comparable efficacies in the inhibition of the BR
In some cases, triazole derivatives affect multiple targets;
although usually to a varying extent . To address whether
Pcz inhibition is specific to the BR biosynthetic pathway or also
affects P450 monooxgenases in GA biosynthesis, we compared
coapplications of Pcz with BL or GA3. Again, Arabidopsis seedlings
cotreated with Pcz and BL showed no difference in primary root
length compared to mock conditions (Fig. 3A, 3C). In contrast, Pcz
induced-inhibition of root length was not recovered by exogenous
co-application of GA3 (Fig. 3A, 3C). We did find a slight increase
(17%) in root length with the co-application of Pcz and GA3
relative to Pcz treatment, however, a similar increase (11%) was
found for GA3 application compared to mock. Taken together, the
results indicate that the inhibition of root growth caused by Pcz is
complemented by the co-application of BL, but not GA3.
Arabidopsis brassinazole resistant1-1D (bzr1-1D) mutants carry a
dominant gain of function mutation in the transcription factor and
major component of BR signaling, BZR1, which results in a
constitutive BR response even in the absence of BRs. Therefore,
bzr1-1D mutants are sensitive indicators for BR inhibitor specificity
. Wild-type (Col-0) plants treated with 2 mM Pcz showed a
reduction in hypocotyl length of 83%, whereas bzr1-1D mutants
exhibited only 4% shorter hypocotyls, compared to mock (Fig. 3D
F). Although bzr1-1D plants were more resistant to Brz
applications than wild type, 2 mM Brz did reduce the length of bzr1-1D
hypocotyls by 37% compared to mock (Fig. 3EF).
BR biosynthesis mutants show reduced sensitivity
The azole ring of Brz binds to the heme prosthetic group of DWF4
(CYP90B1), the rate-limiting enzyme in BR biosynthesis, forming a
coordination complex, which impairs DWF4 activity . Mutants
illustrated (n.15). (D) 3-day old Ws-2 seedlings were transferred to K
MS media containing 1 mM of Pcz, Brz, Ucz, or co-applications of
inhibitors (1 mM) with 0.1 mM BL or 0.1 mM BL alone and incubated for 4
more days. Average root lengths at the end of treatment (n.15). (CD)
Error bars represent standard deviation and lowercase letters indicate
significant differences among treatments determined by Post-hoc
test. Scale bar (A) 1 cm and (B) 0.5 cm.
already deficient in BR biosynthesis should thus show a reduced
sensitivity towards BR biosynthetic inhibitors such as Brz and Pcz.
We tested wild-type and dwf7-1 seedlings with Pcz and compared
their responses to Brz treatments (Fig. 4). DWF7 catalyzes the
conversion of episterol to 5-hydroepisterol upstream of DWF4 .
Wild-type (Ws-2) plants treated with either 1 mM Pcz or Brz, or
10 mM Brz produced 46%, 37%, and 64% shorter roots compared
to mock conditions, respectively (Fig. 4A, 4F). Although dwf7-1
showed a significant response to 1 mM Pcz and 10 mM Brz
treatments, the relative reduction (25% and 30%, respectively)
was still lower than Pcz/Brz treated Ws-2 (Fig. 4B, 4G). bri1-5, a
weak allele of the major BR receptor BRASSINOSTEROID
RESISTENT1 (BRI1), and wild type respond similarly to Brz, but
not to BL treatment . This suggests that although bri1-5 is
affected in BR signal transduction, it still responds to changes in BR
homeostasis. We found that Pcz and Brz, at equal concentrations,
had similar effect on bri1-5 and Ws-2 (Fig. 4A, 4C, 4F, 4H).
Since DWF4 has been shown to be a direct target of Brz ,
which is structurally similar to Pcz (Fig. 1), we also took a closer
look at the effect of Pcz on dwf4 mutants. Ws-2 plants again
showed a decrease in root length upon Pcz or Brz treatment
(Fig. 5A, 5C), but dwf4-1 mutants did not exhibit a significant
reduction (Fig. 5B, 5C).
Pcz specifically influences the expression of BR
Transcriptional feedback regulation of rate-limiting step
enzymes is a critical mechanism to maintain hormone homeostasis
. Since triazole derivatives can have multiple targets, we
evaluated the expression levels of both BR (DWF4, BR6ox2, CPD,
BAS1 and BZR1) and GA (GA20ox1 and GA2ox1) related genes, to
further assess the specificity of Pcz.
Using quantitative real-time PCR (qRT-PCR) we found a
dosedependent induction of BR biosynthetic gene expression (DWF4,
BR6ox2, and CPD) with increasing Pcz concentrations (Fig. 6A).
With the same treatment conditions the mRNA levels of BAS1, a
gene involved in BR degradation , showed a downward trend
(Fig. 6B). BZR1, a key regulator of BR signaling, did not show
relevant changes in its expression upon Pcz treatments (Fig. 6B).
The GA biosynthesis gene GA20ox1, which is feedback regulated
by endogenous GA levels , also showed no increase in
expression upon Pcz application (Fig. 6B).
The Pcz-induced increase in mRNA accumulation of DWF4 and
BR6ox2 and concomitant decrease in BAS1 expression was offset by
co-application of Pcz with BL, but not by Pcz with GA3 (Fig. 6C).
Our results also showed an expected decrease in DWF4 and BR6ox2
expression upon BL and to a smaller extent GA20ox1 upon GA3
treatment (p = 0.06) (Fig. 6CD). Together these findings suggest
that Pcz specifically affects BR regulated genes and do not provide
evidence for an inhibitory effect on GA biosynthesis.
Pcz induces dwarfism in dark and light grown maize
inbred W22 seedlings
Studies on crop species such as sorghum, rice, and maize often
require large amounts of growth media which limits the use of cost
intensive inhibitors such as Brz. For that reason, we tested the effect
of Pcz on both dark and light grown seedlings of the maize inbred
line W22. A strong reduction of hypocotyl elongation is one of the
most striking characteristics of the de-etiolation phenotype of dark
grown Arabidopsis mutants deficient in BR biosynthesis .
Compared to mock, W22 seedlings grown in the dark for 8 d in
the presence of 0.5 to 30 mM Pcz showed decreased mesocotyl
elongation ranging from 27% to 64%, respectively (Fig. 7A, 7C).
Ucz treatment of 0.5 to 30 mM reduced the length of the mesocotyl
25% to 73% (Fig. 7C). Similar to the mesocotyl, the lengths of true
leaves were reduced from 21% to 51% by Pcz and from 20% to 56%
by Ucz, respectively (Fig. 7D). Although both Pcz and Ucz treatment
affected the coleoptile, the relative reduction in length was less
pronounced than in mesocotyls and true leaves (Fig. 7E).
Surprisingly, the primary root of W22 seedlings showed significant
differences in sensitivity towards Pcz and Ucz. Pcz concentrations
up to 30 mM had no significant effect on primary root length. In
contrast, seedlings treated with equal or greater than 5 mM Ucz
resulted in significantly shorter primary roots when compared to
mock (Fig. 7F), reaching a reduction of 73% at 30 mM.
Pcz treatment also induced dwarfism in light grown W22
seedlings. Plants treated with 0.2 to 5 mM Pcz decreased their
overall height by 29% to 45%, respectively (Fig. 7B, 7G). In
addition to the dwarf stature, Pcz induced shorter leaves and
resulted in a more overall compact appearance (Fig. 7B).
Interestingly, unlike in dark treatments, the primary root length
of seedlings grown for 21 d in the light was reduced by 30% when
treated with 5 mM Pcz relative to mock (Fig. 7H).
The genetic background of maize influences tissue
specific sensitivity to Pcz
Given the great diversity between maize inbred lines , we
assessed whether the genetic background influences the effects of
Pcz and Ucz on dark grown maize seedlings. Thus, we repeated
the dark assay using 3 additional maize inbred lines: Mo20W,
A619, and B73. The length of the four evaluated tissues
(mesocotyl, true leaves, coleoptile, and primary root) showed
significant differences between Mo20W, A619, and B73 even in
the absence of Pcz or Ucz (Fig. 8AG). In addition, when treated
with Pcz or Ucz we observed significant differences between the
inbred lines in both their overall and tissue-specific sensitivity. In
the presence of 1 or 10 mM Pcz the mesocotyl length of Mo20W
was reduced by 57% and 56%, respectively, relative to mock
treatment (Fig. 8A, 8D). Likewise, B73 treated with the same
concentrations of Pcz exhibited 40% and 52% shorter mesocotyls,
respectively (Fig. 8CD). However, in the case of A619, only
10 mM Pcz had a significant but smaller impact (35%) on
mesocotyl elongation, relative to mock (Fig. 8B, 8D). Comparable
results were obtained for the response of true leaves to Pcz and
Ucz treatments (Fig. 8AC, 8E).
The primary roots of Mo20W, A619, and B73 showed a
significant reduction in length when treated with 1 or 10 mM Ucz
(Fig. 8AC, 8G). Interestingly, Mo20W and B73 showed, in addition
to the Ucz sensitivity, a decrease in primary root length when treated
with 1 or 10 mM Pcz (Fig. 8A, 8C, 8G). The primary root length of
A619 was reduced significantly only at 10 mM Pcz, but to a smaller
extent than Mo20W and B73 (Fig. 8AC, 8G). The coleoptile was
the only tissue evaluated with comparable responses to both Pcz and
Ucz in all three inbred lines (Fig. 8AC, 8F).
To test whether the differences in Pcz efficacy are due to
differences in BR response, we compared the effect of BL on root
elongation between the maize inbreds. W22 and A619 plants
treated with 20 mM BL exhibited the smallest relative reduction
(37% and 44%, respectively) in root length (Fig. 9). On the other
hand, Mo20W and B73 inbreds treated with the same amount of
BL had 67% and 60% shorter roots than mock, respectively
(Fig. 9). This result is consistent with our previous findings for Pcz
sensitivity, and supports our hypothesis that genetic diversity
influences BR responses in maize.
Figure 5. Resistance of dwf4-1 to Pcz and Brz. (AB) Seedlings of (A) Ws-2 and (B) dwf4-1 grown on K MS media for 7 days, and then
transferred to media containing 1 mM Pcz or Brz for 3 more days of growth. (C) Average root lengths of Ws-2 and dwf4-1 measured at the end of
treatments (n.10). Error bars represent standard deviation and lowercase letters indicate significant differences among treatments determined by
Post-hoc test (p,0.05). Scale bar (AB) 1 cm.
Figure 6. Pcz impact on the expression of BR- and GA-related genes. (AD) Quantitative real-time PCR analysis of the transcript levels after
treatment with 0.2, 1, 5 or 10 mM Pcz of (A) BR biosynthetic genes DWF4, BR6ox2, and CPD or (B) BAS1, BZR1, and GA20ox1, involved in BR
degradation, BR signaling and GA biosynthesis, respectively. (C, D) Expression pattern of (C) BR biosynthesis and signaling or (D) BR degradation, GA
biosynthesis and GA degradation (GA2ox1) genes measured after treatments with 1.5 mM Pcz, 0.01 or 1 mM BL, or 1 mM GA3 or co-applications of Pcz
(1.5 mM) with 0.01 or 1 mM BL or 1 mM GA3. (AD) Data points represent the average of three independent biological replicates with three technical
replicates each. Error bars represent standard deviation. Asterisks indicate significant differences to the respective mock determined by Students
ttest (p,0.05). Ubiquitin conjugating enzyme 21 (UBC21) was used as internal control.
Phytohormone biosynthesis inhibitors allow the
species-independent study of hormonal function during plant development.
Inhibitor studies can also support the isolation and
characterization of hormone deficient mutants without prior knowledge of the
mutant phenotype. Pcz has previously been reported to impair the
hypocotyl growth of cress seedlings and that this inhibition is
reversible by the co-application of BL . Chemical modification
of Pcz also revealed structural elements essential for its inhibitory
properties . Pczs high accessibility and economical aspects
prompted us to conduct a comparative analysis with the
established BR inhibitor Brz.
Pcz treatment of Arabidopsis seedlings produced typical
BRdeficient phenotypes such as: epinastically growing and dark-green
cotyledons, reduced hypocotyl length, and a significantly shorter
primary root (Fig. 2). Using root length as a reference we found that
even relatively low Pcz levels of 0.5 mM resulted in strong inhibition
(Fig. 2). As shown in independent experiments, the impairment of
root growth in Arabidopsis through Pcz treatment can essentially be
restored to length of mock-treated seedlings by BL, but not GA3
(Fig. 3). The slight effect of GA3 on root elongation was independent
of Pcz treatment and may not indicate a recovery of Pcz inhibition.
In contrast, BL treatment had a dramatic effect on root elongation in
Pcz treated seedlings (Fig. 3).
In the absence of BR, the transcription factor BZR1 and its
homolog BZR2 (BES1) are phosphorylated by the GSK3/
SHAGGY-like protein kinase BIN2 [49,5556]. Phosphorylation
negates BZR1s DNA-binding capacity and increases its
cytoplasmic retention by phosphopeptide-binding 14-3-3 proteins .
The dominant bzr1-1D mutation increases BZR1s
dephosphorylation by the phosphatase PP2A . BZR1 therefore remains
nuclear localized and stabilized even in the absence of BRs causing
bzr1-1D plants to show a constitutive BR response [49,59]. In
contrast to wild type, bzr1-1D mutants showed only a minor
inhibition of hypocotyl growth in the presence of Pcz (Fig. 3).
Current evidence in rice and cress suggests that Brz inhibits BR
biosynthesis but also affects GA responses . We found that
bzr1-1D plants were more sensitive to Brz than Pcz (Fig. 3) which
suggests that Pcz is more specific. This hypothesis is supported by
our finding that roots co-treated with Brz and BL, but not with Pcz
and BL, were significantly shorter than mock (Fig. 2).
Ucz has been extensively studied as an inhibitor of GA
biosynthesis . Circumstantial evidence reported by Yokota et
al.  and Iwasaki and Shibaoka  indicates that Ucz might
also act as a demethylase inhibitor in BR biosynthesis. We
observed shorter roots and hypocotyls of Ucz-treated Arabidopsis
seedlings. Although Pcz- and Ucz-induced phenotypes were
similar, co-application of Ucz and BL was not significantly
different to Ucz alone (Fig. 2). Based on these analyses,
Pczmediated suppression of root and hypocotyl elongation is likely the
result of a specific inhibition of the BR biosynthetic pathway.
DWF7/STE1 is a D7 sterol C-5 desaturase that converts
avenasterol to dehydroavenasterol or episterol to
5-dehydroepisterol early in BR biosynthesis . Significant reduction of root
elongation with Pcz treatment was found in wild type and to lesser
extent also for dwf7-1 seedlings, whereas roots of dwf4-1 mutants
did not exhibit a significant decrease (Fig. 4, Fig. 5). The BR
metabolic pathway is likely non-linear, as downstream BR
intermediates can be found in most monogenic BR biosynthetic
mutants, including dwf7-1 [18,20,60]. Therefore, Pcz treatment
may further reduce endogenous BR pools in dwf7-1 mutants. Loss
of function mutations in DWF4 result in a more severe phenotype
which could be the reason why no further reduction in root length
was observed in dwf4-1 upon Pcz treatment. Alternatively, since
DWF4 could be the target of Pcz like for Brz  an additional
inhibition of growth can not be expected in an already genetically
disrupted dwf4-1 mutant. From our findings we conclude that BR
biosynthesis mutants show a reduced sensitivity towards Pcz.
Another line of evidence that Pcz is a specific and potent BR
biosynthesis inhibitor comes from transcriptional analyses of BR
and GA regulated genes in Arabidopsis. BR homeostasis relies on
the feedback regulation of DWF4 transcription . Thus,
differences in DWF4 expression reflect even minor changes in
BR biosynthesis. As expected, we found that BL treatment
reduced the expression of DWF4 and other BR-biosynthetic genes
in wild type, whereas Pcz application resulted in a dose-dependent
increase of DWF4, CPD and BR6ox2 transcripts (Fig. 6). The
induction of CPD expression relative to DWF4 and BR6ox2 was
lower upon Pcz application, however, CPD is primarily
posttranscriptionally regulated . The Pcz dependent induction of
BR biosynthetic gene expression was offset by the co-application of
BL similar to BL treated controls. With co-application of GA3 this
Pcz dependent induction was not reverted (Fig. 6).
PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1/
CYP72B1) catalyzes the conversion and inactivation of BL to
26Hydro-BL. Similar to DWF4, BAS1 is feedback regulated by
endogenous BR levels . We observed that BAS1 expression was
induced by BL application and repressed by Pcz treatment
(Fig. 6).The co-application of Pcz with BL, but not with GA3,
countered the effect of Pcz on BAS1 repression.
GA20-oxidases catalyze the sequential conversions of GA53 to
GA20, late in the GA biosynthetic pathway . Similar to DWF4,
GA20ox-metidated steps are flux determining  and their
expression is under feedback regulation . Similar to the
observations for BR-biosynthetic genes, GA20ox1 expression
appeared negatively regulated by GA3 (Fig. 6). However, mRNA
levels of GA20ox1 were not increased over a broad spectrum of Pcz
concentrations (Fig. 6). These findings are corroborated by data
from the Genevestigator database , which also shows a
regulation of GA20-oxidases by both GA and Pac, but not Pcz.
The expression pattern of GA catabolic gene GA2ox1  showed
an expected induction upon GA3 application, but did not display
relevant differences with either Pcz or BL treatments (Fig. 6).
Interestingly, the GA-dependent increase in GA2ox1 expression
was impaired by simultaneous treatment with Pcz (Fig. 6).
Interpreting this result, we cannot exclude the possibility of a
BR-dependent GA regulation of GA2ox1 expression. Our data also
showed an overlap in the expression patterns of BAS1 and
GA20ox1 (Fig. 6). Taken together these results do not provide
evidence for a negative effect of Pcz on GA biosynthesis.
To investigate if Pcz responses found in Arabidopsis can be
corroborated with monocot plants we chose maize, a member of
the prominent grass family (Poaceae). This family, of close to 10,000
species, encompasses important genetic models like Brachypodium
distachyon , as well as important food crops such as wheat, rice,
and maize. Recently, we have shown that Pcz treatment of wild
type maize phenocopies the BR deficient dwarf nana plant1 (na1)
and also that na1 plants are more Pcz resistant than wild type or
GA impaired mutants . Using comparative treatments of
increasing Pcz or Ucz concentrations we found a strong decrease
in the mesocotyl length of dark-grown W22 seedlings (Fig. 7).
Similar responses were detected for true leaves (Fig. 7). In
comparison, the response of coleoptiles towards inhibitor
treatments was less pronounced (Fig. 7). This indicates either a
tissuespecific sensitivity towards Pcz or different BR levels in coleoptiles.
The coleoptile, whose main role is support of juvenile leaves
during soil penetration, originates directly from the pro-embryo
and not from the apical meristem like the true leaves. It is
therefore possible that these tissues have different reception and
signaling systems for BRs.
In contrast to the results obtained with Arabidopsis we discovered
that W22 roots were resistant towards Pcz. While Ucz treated
dark-grown roots of W22 showed drastically reduced elongation,
no significant response was observed over a broad range of Pcz
concentrations up to 30 mM (Fig. 7). While light-grown maize
seedlings are obligate heterotrophic until day 7, an equal balance
between heterotrophic and autotrophically produced carbon is
reached on day 10 for leaves, and day 13 to 14 for roots . The
slight reduction (30%) in root length observed in light-grown
seedlings may be explained by the fact that these plants were
measured after the switch to autotrophy when most of their carbon
comes from photosynthesis. The strong reduction in plant height
and decrease in photosynthetically active leaf surface at 5 mM Pcz
treatment suggests that the plants had a decreased capacity to
produce photosynthates. On the other hand, we analyzed
darkgrown plants during a phase when they received most nutrients
from the endosperm. These results may allude to fundamental
differences in the control of cell elongation between W22 and
Maize roots contain the enzymes for the late C-6 oxidation steps
of BR biosynthesis . Our observation of differential Pcz
resistance of W22 roots raised the question if this is a feature
specific to W22 inbreds. We therefore tested the effect of genetic
diversity in maize inbreds  towards Pcz response using the
lines Mo20W, A619, and B73. Significant differences between
these inbred lines in the length of four analyzed tissues were
observed even under mock conditions. Furthermore, we found
significant differences in tissue specific sensitivity towards Pcz and
Ucz (Fig. 8). In general, Mo20W showed the highest sensitivity
and A619 the highest resistance towards both inhibitors.
Concerning tissue-specific responses, the coleoptile was the only
organ which showed an even response to both Pcz and Ucz
treatment in Mo20W, A619, and B73. In contrast, Pcz sensitivity
in the roots and true leaves ranged from resistant (A619) to highly
susceptible (B73 and Mo20W). The degree of Pcz response in
maize roots seems therefore dependent on the genetic background
of the maize line. The data also indicates differential hormonal
regulation of tissue growth in aerial organs of maize inbreds. In
rice and wheat tissue culture, accumulation of Pcz against a
concentration gradient has been reported . This indicates
active uptake systems in these grass species. In Monilinia fructicola,
the ABC transporter MfABC1 is induced upon Pcz treatment,
which suggests a possible role for transporters of the ABC family
 in Pcz uptake in plants and fungi . Differences in either
root uptake, in planta transport, and/or Pcz catabolism may be
responsible for the observed variances between maize inbreds.
Nonetheless, our results also indicated a relation of Pcz- and
BLsensitivity between the inbred lines. Compared to Mo20W and
B73, W22 and A619 plants exhibited a smaller inhibition of root
elongation in the presence of either Pcz or higher concentrations
of BL (Fig. 8, Fig. 9). We therefore conclude that the genetic
diversity between these maize lines influences their response to
We presented independent lines of evidence which indicate that
Pcz inhibits BR metabolism and induces BR deficiencies in both
Arabidopsis and maize seedlings. Arabidopsis seedlings treated with
Pcz phenocopied BR deficient mutants such as dwf7 and dwf4.
Similarly, Pcz-induced dwarf phenotypes were discovered in both
light and dark grown maize seedlings. Growth responses towards
Pcz and Ucz were not equally expressed in all measured tissues of
maize. Tissue specific sensitivity of Pcz in the coleoptile,
mesocotyl, true leaves, and primary roots alludes to differential
BR biosynthesis and/or signal transduction for the different maize
tissues. Genetic variation of maize inbred lines implies that genetic
enhancers and suppressors play a key role in Pcz-induced
physiological responses. We presented that Pcz is a potent
alternative to the commonly used Brz with a comparable
specificity and efficacy. In contrast to Brz/Brz2001, Pcz is easily
accessible and the associated costs are much lower, allowing its use
for large-scale chemical genomics and field testing.
Materials and Methods
Plant material and growth conditions
Seeds of Arabidopsis thaliana were surfaced-sterilized before being
sprinkled on 0.8% agar-solidified media containing 0.56
Murashige and Skoog salts and 1% sucrose. After one day of
stratification at 4uC, plates were transferred to a growth room
and grown under a 16 h photoperiod. For the bzr1-1D
experiments, seedlings were stratified for 48 h at 4uC, irradiated for
6 hours to promote germination and then transferred to a growth
chamber and grown in the dark at 22uC.
Maize plants were grown under greenhouse conditions at 27uC
(day) and 21uC (night). Unless indicated otherwise, plants were
grown in coarse Vermiculite (SunGro Horticulture, Bellevue, WA
and Perlite Vermiculite Packaging Industries, Inc., North
Bloomfield, OH). Plants were fertilized with 200 ppm Miracle-Gro Excel
(Scotts, Marysville, OH) adjusted to pH 6 following manufacturer
Chemical treatments and morphometric analysis
Seedlings of 3-day old Ws-2 wild type were transferred to
agarsolidified media supplemented with Pcz (Banner Maxx, Syngenta,
Greensboro, NC), Brz (gift from Shozo Fujioka, Riken, Japan) and
Ucz (Consise, Fine Americas Inc., Walnut Creek, CA) alone or in
combination with BL (Sigma Aldrich, St Louis, MO) or GA3 (Gold
Biotechnology, St. Louis, MO). Media plates were placed
vertically to ease morphometric analysis of the root and each
plate contained more than 10 seedlings. After 3 days of treatment,
images were taken and the growth parameters were analyzed using
ImageJ software . For treatments with Pcz and Brz of BR
mutants (dwf4-1, dwf7-1 and bri1-5) and its Ws-2 wild type, the
seedlings were grown for 7 days on MS media before being
transferred to the inhibitor-containing media. Measurements were
done after 3 days of the treatment. bzr1-1D and its wild type Col-0
seedlings were grown in the dark on MS media containing Pcz or
Brz for 7 days and hypocotyl lengths were measured.
For all treatment experiments in maize, seeds were sterilized for
7 min at 60uC in a water bath prior to planting and grown under
greenhouse conditions. For de-etiolation assays, maize seeds were
imbibed for 28 h in paper towels and soaked with distilled water
containing indicated concentrations of Pcz or Ucz. They were
then planted 10 cm deep in 15 cm wide pots with coarse
Vermiculite, watered with identical concentrations of Pcz or
Ucz, and grown for additional 7 d at 28uC and 90% humidity in
the dark. Control plants were grown in the dark or light in the
absence of Pcz or Ucz treatment. Plants were then harvested,
photographed, and analyzed using ImageJ software .
Mesocotyl length was determined from the root-shoot transition zone to
the first node. Coleoptile and true leaf length was measured from
the first node to the tip of the coleoptile or true leaves, respectively,
whereas the length of the main root was used to determine root
For light grown Pcz experiments, W22 seeds were planted 5 cm
deep in 24.5 cm wide pots with coarse Vermiculite and watered
every fifth day. Pcz was added at indicated concentrations to the
water solution. After 21 days plants were harvested, photographed,
and analyzed using ImageJ . Plant height was measured from
the root-shoot transition zone to the highest leaf collar, whereas
the length of the main root was used to determine root length.
RNA extraction and qRT-PCR detection of gene
Arabidopsis thaliana Ws-2 seeds were surface sterilized and
stratified for 48 h at 4uC, followed by growth for 4 days at
100 mmol/m2/sec, 16:8 h light/dark cycle at 25uC. The seedlings
were then transferred into Erlenmeyer flasks prefilled with 50 ml
K MS with 1% sucrose liquid media (pH 5.7) and were allowed to
grow for 2 d at 100 rpm under the conditions described above.
For the treatments, 5006 stock solutions were made in 50%
DMSO (0.1% final concentration) and 100 ml of each stock
solution, or 50% DMSO for the mock treatment, were applied at
the beginning of the light cycle on day 7. After 10 h at 100 rpm
the seedlings were harvested and immediately frozen in liquid
Total RNA was isolated from seedlings, as described by
Eggermont et al. . For qRT-PCR analysis, total RNA was
pre-treated with DNase I (Invitrogen), and cDNA was synthesized
using Reverse Transcriptase (Invitrogen). Ubiquitin conjugating
enzyme 21 used as internal control was amplified with
UBC21_FOR (300 nM) and UBC21_REV (300 nM). Gen-specific primers
used were: DWF4_FOR1 (500 nM); DWF4_REV1 (500 nM);
BR6ox2_FOR1 (300 nM) and BR6ox2_REV1 (300 nM);
CPD_FOR1 (500 nM) and CPD_REV1 (500 nM); BAS1_FOR1
(1100 nM) and BAS1_REV1 (1100 nM); BZR1_FOR1 (300 nM)
and BZR1_REV1 (300 nM); GA2ox1_FOR2 (500 nM) and
GA2ox1_REV2 (500 nM) as well as GA20ox1_FOR1 (500 nM)
and GA20ox1_REV1 (500 nM). Primer sequences are listed in
Table S4. All primers showed .90% efficiency at their indicated
concentrations. qRT-PCRs were performed as described
previously [23,74] using the StepOnePlus instrument (Invitrogen). Each
data point represents the average of three independent biological
replicates (approximately 30 samples per replicate), with three
The Microsoft Excel add-in XL Toolbox (ver. 3.02, http://
xltoolbox.sourceforge.net) was used to obtain all descriptive and
comparative statistics. Analyses of variance (ANOVA) for sets of
data groups were performed with Multiple
comparisons/Posthoc testing. Once a significant difference (p,0.05) was detected,
Post-hoc tests, using the Holm-Sidak algorithm, were performed
to test which of the possible multiple comparisons between the
data groups were significant .
Table S1 Statistical analysis of Figure 7 CF. Statistic
analysis was performed using ANOVA with Post Hoc test using
the Holm-Sidak algorithm. Adjusted a and adjusted p-values are
shown and significance of p-values was indicated with bold text.
Table S2 Statistical analysis of Figure 8 DG. Statistic
analysis was performed using ANOVA with Post Hoc test using
the Holm-Sidak algorithm. Adjusted a and adjusted p-values are
shown and significance of p-values was indicated with bold text.
Table S3 Statistical analysis of Figure 9. Statistic analysis
was performed using ANOVA with Post Hoc test using the
Holm-Sidak algorithm. Adjusted a and adjusted p-values are
shown and significance of p-values was indicated with bold text.
Table S5 Number of visible leaves and leaf collars of
Pcz treated W22. W22 maize seedlings grown in the light for 3
weeks in the presence of 0, 0.2, 1, or 5 mM Pcz. All visible,
including immature leaves of treated plants (n.13) were counted.
The leaf collar was recorded if a ligule was developed. Students
We thank A. Klempien, R. Altstatt, M. Gutensohn, and R. Weizbauer for
critical reading of the manuscript and Shozo Fujioka (RIKEN) for the
generous gift of brassinazole. We are grateful to Zhiyong Wang (Carnegie
Institution) for support and discussion of data.
Conceived and designed the experiments: TH CC SC BS. Performed the
experiments: TH CC NBB JSB JYZ. Analyzed the data: TH CC NBB JSB
JYZ SC BS. Wrote the paper: TH CC NBB JSB SC BS.
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