Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize

Abstract

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.

About the Authors:

Thomas Hartwig

Contributed equally to this work with: Thomas Hartwig, Claudia Corvalan

Affiliation: Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America

Claudia Corvalan

Contributed equally to this work with: Thomas Hartwig, Claudia Corvalan

Affiliation: School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea

Norman B. Best

Affiliation: Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America

Joshua S. Budka

Affiliation: Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America

Jia-Ying Zhu

Affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, California, United States of America

Sunghwa Choe

* E-mail: [email protected] (SC); [email protected] (BS)

Affiliations School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea, Plant Genomics and Breeding Institute, Seoul National University, Seoul, Korea

Burkhard Schulz

* E-mail: [email protected] (SC); [email protected] (BS)

Affiliation: Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America

Introduction

Brassinosteroids (BRs) are poly-hydroxylated steroidal hormones with profound effects on several physiological plant responses. They are involved in regulating cell elongation and division [1]–[2], vascular differentiation [3]–[4], photomorphogenesis [5]–[6], leaf angle inclination [7]–[8], seed germination [9]–[10], stomata development [11], as well as suppression of leaf senescence and abscission [12]. Radioactive tracer studies in cell cultures of Catharanthus roseus established the steps of the BR metabolic pathway [13]. 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 [21]. 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 [22], only a few null-mutations have been reported in these species [23]–[24].

The field of chemical genomics greatly benefited from the use of chemical inhibitors/modifiers [25]–[26]. 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]–[28].

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 [29]. 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 [30]. Both compounds inhibit P450 CYP701, which catalyzes an early step in GA biosynthesis [30]. Furthermore, Ucz also has been reported to slightly decrease the endogenous concentration of castasterone and inhibits BR-induced tracheary element differentiation [31]–[32]. 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 [33]–[34].

[Figure omitted. See PDF.]

Figure 1. Chemical structures of brassinazole, propiconazole, paclobutrazole, and uniconazole.

Structure elements critical for inhibitor activity have been color-coded: (blue) nitrogen atoms in the azole ring; (purple) chlorine atom(s) of the phenyl ring; and (red) either primary/secondary hydroxyl group or 1,3-dioxlane. Structures were drawn using the ChemBioDraw 12.0.2 software and structures were compared to the ChemACX 12.12.1 database.

https://doi.org/10.1371/journal.pone.0036625.g001

Brz's mode of action is to bind and block DWF4/CYP90B1 [35]. DWF4 is a P450 that mediates multiple rate-limiting C22 alpha-hydroxylations in the biosynthesis of BRs [35]. DWF4 expression is a target of regulation by both endogenous signals like auxin [36]–[37] and exogenous cues like temperature [38]. Brz and its even more specific derivative Brz2001 [39] became invaluable tools for BR research. Not only did they help to reveal the role of BRs in various plant species [40], they were also essential for the isolation and characterization of genes that function in BR signaling [41]–[43]. 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-[ [2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1,2,4-triazole, (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) [27]. 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-(4-trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole, were identified [27]. 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) [44]–[45]. Pcz has also been studied extensively for its toxicity on plants, animals, humans, and the environment [46]–[47]. Here we present a molecular genetic analysis of Pcz's effects on Arabidopsis and maize seedlings. Our results indicate that Pcz is a potent and specific inhibitor of the BR metabolic pathway in plants.

Results

Arabidopsis seedlings treated with Pcz display dwarf phenotypes

To study Pcz's impact on Arabidopsis, we treated wild-type Ws-2 plants with Pcz concentrations ranging from 0.1 to 5 µM for 5 days. Cotyledons showed a reduction in size and epinatic growth responses with treatments of Pcz (Fig. 2A–B). 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 µM Pcz decreased the elongation by 20% and 5 µM Pcz by 54% compared to mock conditions (Fig. 2A, 2C). No further significant decrease was observed at concentrations higher than 0.5 µM Pcz (Fig. 2C).

[Figure omitted. See PDF.]

Figure 2. Responses of Arabidopsis seedlings to inhibitor treatments and BL Complementation.

(A–C) 3-day old Ws-2 seedlings were transferred to ½ MS media containing 0, 0.1, 0.5, 1, or 5 µM of Pcz and incubated for 5 more days. (A) Seedlings at the end of treatment (5 d). (B) Close-up of the cotyledons and true leaves in the same order as (A). (C) Average root lengths at day 3 of treatment are illustrated (n>15). (D) 3-day old Ws-2 seedlings were transferred to ½ MS media containing 1 µM of Pcz, Brz, Ucz, or co-applications of inhibitors (1 µM) with 0.1 µM BL or 0.1 µM BL alone and incubated for 4 more days. Average root lengths at the end of treatment (n>15). (C–D) 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.

https://doi.org/10.1371/journal.pone.0036625.g002

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 µM 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 µM 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 µM Pcz or Brz, but not 1 µM Ucz, with co-applications of 0.1 µM 24epi-brassinolide (BL), a bioactive epimer of brassinolide (Fig. 2D). Root length of plants co-treated with 1 µM Pcz plus 0.1 µM BL was not significantly different from mock treatments (Fig. 2D). On the contrary, the co-application of 1 µM Ucz and 0.1 µM BL showed no significant difference to treatments with 1 µM Ucz alone (Fig. 2D). These results suggest that Pcz and Brz have comparable efficacies in the inhibition of the BR biosynthetic pathway.

In some cases, triazole derivatives affect multiple targets; although usually to a varying extent [48]. To address whether Pcz inhibition is specific to the BR biosynthetic pathway or also affects P450 monooxgenases in GA biosynthesis, we compared co-applications of Pcz with BL or GA3. Again, Arabidopsis seedlings co-treated 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.

[Figure omitted. See PDF.]

Figure 3. Pcz specificity towards BR biosynthesis inhibition.

(A–C) 3-day old Ws-2 were transferred to ½ MS media containing either 1 µM Pcz, 0.1 µM BL, 10 µM GA3, or co-applications of 1 µM Pcz with 0.1 µM BL or 10 µM GA3 and grown for 3 more days. (A) Seedlings at the end of treatment. (B) Close-up of the cotyledons and true leaves in the same order as (A). (C) Average root lengths at day 3 of treatment (n>15). (D–F) Col-0 and bzr1-1D (Col-0) grown on ½ MS media in the dark for 7 days. (D) Col-0 or (E) bzr1-1D grown on medium with 2 µM Pcz or 2 µM Brz. (F) Hypocotyl lengths of Col-0 and bzr1-1D grown on medium with 2, 4, 8 µM Pcz or 2 µM Brz (n≥10). (C, F) Error bars represent standard deviation. (C) Lowercase letters indicate significant differences among treatments determined by “Post-hoc” test (p<0.05). (F) Asterisks indicate significant difference to the respective mock determined by Student's t-test (p<0.01). Scale bar (A, D, E) 1 cm and (B) 0.5 cm.

https://doi.org/10.1371/journal.pone.0036625.g003

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 [49]. Wild-type (Col-0) plants treated with 2 µM 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 µM Brz did reduce the length of bzr1-1D hypocotyls by 37% compared to mock (Fig. 3E–F).

BR biosynthesis mutants show reduced sensitivity towards Pcz

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 [35]. Mutants 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 [18]. Wild-type (Ws-2) plants treated with either 1 µM Pcz or Brz, or 10 µM 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 µM Pcz and 10 µM 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 [50]–[51]. 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).

[Figure omitted. See PDF.]

Figure 4. Sensitivity of Arabidopsis wild type, BR biosynthetic mutant dwf7-1, and signaling mutant bri1-5 to Pcz and Brz.

(A–C) Seedlings of (A) Ws-2, (B) the BR biosynthetic mutant dwf7-1 (Ws-2), and (C) the BR signaling mutant bri1-5 (Ws-2) grown on ½ MS media with either 1 µM Pcz or Brz or 10 µM Brz. (D–E) Close-up of the shoot apices harboring cotyledons and the first pair of true leaves of (D) dwf7-1 and (E) bri1-5. (F–H) Average root lengths of (F) Ws-2, (G) dwf7-1 and (H) bri1-5 measured at the end of treatments (n>10). (F–H) Error bars represent standard deviation and lowercase letters indicate significant differences among treatments determined by “Post-hoc” test (p<0.05). Scale bar (A–E) 1 cm.

https://doi.org/10.1371/journal.pone.0036625.g004

Since DWF4 has been shown to be a direct target of Brz [35], 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).

[Figure omitted. See PDF.]

Figure 5. Resistance of dwf4-1 to Pcz and Brz.

(A–B) Seedlings of (A) Ws-2 and (B) dwf4-1 grown on ½ MS media for 7 days, and then transferred to media containing 1 µM 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 (A–B) 1 cm.

https://doi.org/10.1371/journal.pone.0036625.g005

Pcz specifically influences the expression of BR biosynthetic genes

Transcriptional feedback regulation of rate-limiting step enzymes is a critical mechanism to maintain hormone homeostasis [52]–[53]. 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 dose-dependent 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 [5], 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 [30], also showed no increase in expression upon Pcz application (Fig. 6B).

[Figure omitted. See PDF.]

Figure 6. Pcz impact on the expression of BR- and GA-related genes.

(A–D) Quantitative real-time PCR analysis of the transcript levels after treatment with 0.2, 1, 5 or 10 µM 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 µM Pcz, 0.01 or 1 µM BL, or 1 µM GA3 or co-applications of Pcz (1.5 µM) with 0.01 or 1 µM BL or 1 µM GA3. (A–D) 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 Student's t-test (p<0.05). Ubiquitin conjugating enzyme 21 (UBC21) was used as internal control.

https://doi.org/10.1371/journal.pone.0036625.g006

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. 6C–D). 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 [53]. Compared to mock, W22 seedlings grown in the dark for 8 d in the presence of 0.5 to 30 µM Pcz showed decreased mesocotyl elongation ranging from 27% to 64%, respectively (Fig. 7A, 7C). Ucz treatment of 0.5 to 30 µM 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 µM had no significant effect on primary root length. In contrast, seedlings treated with equal or greater than 5 µM Ucz resulted in significantly shorter primary roots when compared to mock (Fig. 7F), reaching a reduction of 73% at 30 µM.

[Figure omitted. See PDF.]

Figure 7. Tissue-specific response of dark and light grown W22 seedlings to Pcz and Ucz.

(A) Maize seedlings grown for 8 d at 29°C in the dark treated with (left to right): 0 µM Pcz, 0 µM Ucz, 1 µM Pcz, 1 µM Ucz, 10 µM Pcz, and 10 µM Ucz, respectively. (B) W22 seedlings grown in the light for 3 weeks at concentrations of (left to right): 0, 0.2, 1, and 5 µM Pcz, respectively. (C–F) W22 seedlings grown for 8 d at 29°C in the dark with Pcz or Ucz at concentrations of 0, 0.5, 1, 5, 10, 20, or 30 µM. Lengths of the (C) mesocotyl, (D) true leaves, (E) coleoptile, and (F) primary root of W22 seedlings grown in the dark with indicated concentrations of Pcz or Ucz (n>15). (G–H) Analysis of W22 maize seedlings grown in the light for 3 weeks at concentrations of 0, 0.2, 1, or 5 µM Pcz (Table S5). (G) Plant height and (H) primary root length was measured (n>15). Error bars represent standard deviation. (C–H) Statistical analysis determined by “Post-hoc” test is shown (C–F) in Table S1 and (G–H) indicated by lowercase letters (p<0.05). Scale bar (A–B) 10 cm.

https://doi.org/10.1371/journal.pone.0036625.g007

Pcz treatment also induced dwarfism in light grown W22 seedlings. Plants treated with 0.2 to 5 µM 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 µM 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 [54], 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. 8A–G). 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 µM 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. 8C–D). However, in the case of A619, only 10 µM 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. 8A–C, 8E).

[Figure omitted. See PDF.]

Figure 8. Impact of the genetic diversity on the tissue specific sensitivity towards Pcz and Ucz.

(A–C) Maize inbred seedlings (A) Mo20W, (B) A619, and (C) B73 grown in vermiculite for 8 d at 29°C in the dark treated with (left to right): 0 µM Pcz, 1 µM Pcz, 10 µM Pcz, 0 µM Ucz, 1 µM Ucz, and 10 µM Ucz, respectively. (D–G) Length of the (D) mesocotyl, (E) true leaves, (F) coleoptile, and (G) primary root of Mo20W, A619, and B73 maize seedlings treated with 0 µM Pcz, 1 µM Pcz, 10 µM Pcz, 0 µM Ucz, 1 µM Ucz, or 10 µM Ucz, respectively (n>15). (D–G) Error bars represent standard deviation. Statistical analysis determined by “Post-hoc” test is shown in Table S2 (p<0.05). Scale bar (A–C) 6 cm.

https://doi.org/10.1371/journal.pone.0036625.g008

The primary roots of Mo20W, A619, and B73 showed a significant reduction in length when treated with 1 or 10 µM Ucz (Fig. 8A–C, 8G). Interestingly, Mo20W and B73 showed, in addition to the Ucz sensitivity, a decrease in primary root length when treated with 1 or 10 µM Pcz (Fig. 8A, 8C, 8G). The primary root length of A619 was reduced significantly only at 10 µM Pcz, but to a smaller extent than Mo20W and B73 (Fig. 8A–C, 8G). The coleoptile was the only tissue evaluated with comparable responses to both Pcz and Ucz in all three inbred lines (Fig. 8A–C, 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 µM 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 omitted. See PDF.]

Figure 9. Influence of the genetic variation on the BR sensitivity in maize root.

Maize inbred seedlings W22, Mo20W, A619, and B73 grown in vermiculite for 8 d at 29°C in the dark in the presence or absences of 1.25, 5, or 20 µM BL, respectively. Average length of the primary root measured at the end of treatment (n>15). Error bars represent standard deviation. Statistical analysis determined by “Post-hoc” test (p<0.05) is shown in Table S3.

https://doi.org/10.1371/journal.pone.0036625.g009

Discussion

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 [27]. Chemical modification of Pcz also revealed structural elements essential for its inhibitory properties [27]. Pcz's 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 BR-deficient 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 µM 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], [55]–[56]. Phosphorylation negates BZR1's DNA-binding capacity and increases its cytoplasmic retention by phosphopeptide-binding 14-3-3 proteins [57]–[58]. The dominant bzr1-1D mutation increases BZR1's dephosphorylation by the phosphatase PP2A [59]. 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 [39]. 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 [30]. Circumstantial evidence reported by Yokota et al. [31] and Iwasaki and Shibaoka [32] 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, Pcz-mediated suppression of root and hypocotyl elongation is likely the result of a specific inhibition of the BR biosynthetic pathway.

DWF7/STE1 is a Δ7 sterol C-5 desaturase that converts avenasterol to dehydroavenasterol or episterol to 5-dehydroepisterol early in BR biosynthesis [18]. 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 [35] – 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 [52]. 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 post-transcriptionally regulated [51]. 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 26-Hydro-BL. Similar to DWF4, BAS1 is feedback regulated by endogenous BR levels [5]. 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 [61]. Similar to DWF4, GA20ox-metidated steps are flux determining [62]–[63] and their expression is under feedback regulation [64]. 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 [65], which also shows a regulation of GA20-oxidases by both GA and Pac, but not Pcz. The expression pattern of GA catabolic gene GA2ox1 [61] 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 [66], 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 [23]. 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 tissue-specific 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 µM (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 [67]. 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 µM Pcz treatment suggests that the plants had a decreased capacity to produce photosynthates. On the other hand, we analyzed dark-grown 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 Arabidopsis roots.

Maize roots contain the enzymes for the late C-6 oxidation steps of BR biosynthesis [68]. 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 [54] 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 [69]. 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 [70] in Pcz uptake in plants and fungi [71]. 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 BL-sensitivity 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 BRs.

Conclusion

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 0.5× Murashige and Skoog salts and 1% sucrose. After one day of stratification at 4°C, 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 4°C, irradiated for 6 hours to promote germination and then transferred to a growth chamber and grown in the dark at 22°C.

Maize plants were grown under greenhouse conditions at 27°C (day) and 21°C (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 recommendations.

Chemical treatments and morphometric analysis

Seedlings of 3-day old Ws-2 wild type were transferred to agar-solidified 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 [72]. 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 60°C 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 28°C 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 [72]. 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 length.

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 [72]. 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 transcripts

Arabidopsis thaliana Ws-2 seeds were surface sterilized and stratified for 48 h at 4°C, followed by growth for 4 days at 100 µmol/m2/sec, 16∶8 h light/dark cycle at 25°C. The seedlings were then transferred into Erlenmeyer flasks prefilled with 50 ml ½ 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, 500× stock solutions were made in 50% DMSO (0.1% final concentration) and 100 µl 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 nitrogen.

Total RNA was isolated from seedlings, as described by Eggermont et al. [73]. 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 technical replicates.

Statistical analysis

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/Post-hoc” 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 [75].

Supporting Information

[Figure omitted. See PDF.]

Table S1.

Statistical analysis of Figure 7 C–F. Statistic analysis was performed using ANOVA with “Post Hoc” test using the Holm-Sidak algorithm. Adjusted α and adjusted p-values are shown and significance of p-values was indicated with bold text.

https://doi.org/10.1371/journal.pone.0036625.s001

(DOC)

Table S2.

Statistical analysis of Figure 8 D–G. Statistic analysis was performed using ANOVA with “Post Hoc” test using the Holm-Sidak algorithm. Adjusted α and adjusted p-values are shown and significance of p-values was indicated with bold text.

https://doi.org/10.1371/journal.pone.0036625.s002

(DOC)

Table S3.

Statistical analysis of Figure 9. Statistic analysis was performed using ANOVA with “Post Hoc” test using the Holm-Sidak algorithm. Adjusted α and adjusted p-values are shown and significance of p-values was indicated with bold text.

https://doi.org/10.1371/journal.pone.0036625.s003

(DOC)

Table S4.

Oligo sequences. Primer sequences used for qRT-PCR as described in Material and Methods. Sequences are listed from 5′ to 3′.

https://doi.org/10.1371/journal.pone.0036625.s004

(DOC)

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 µM Pcz. All visible, including immature leaves of treated plants (n>13) were counted. The leaf collar was recorded if a ligule was developed. Student's t-test was used to obtain the indicated p-values for the comparison with mock.

https://doi.org/10.1371/journal.pone.0036625.s005

(DOC)

Acknowledgments

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.

Author Contributions

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.

Citation: Hartwig T, Corvalan C, Best NB, Budka JS, Zhu J-Y, Choe S, et al. (2012) Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize. PLoS ONE7(5): e36625. https://doi.org/10.1371/journal.pone.0036625

References

1. Azpiroz R, Wu Y, LoCascio JC, Feldmann KA (1998) An Arabidopsis brassinosteriod-dependent mutant is blocked in cell elongation. Plant Cell 10: 219–230.R. AzpirozY. WuJC LoCascioKA Feldmann1998An Arabidopsis brassinosteriod-dependent mutant is blocked in cell elongation.Plant Cell10219230

2. Cheon J, Park SY, Schulz B, Choe S (2010) Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction. BMC Plant Biol 10: 270.J. CheonSY ParkB. SchulzS. Choe2010Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction.BMC Plant Biol10270

3. Yamamoto R, Demura T, Fukuda H (1997) Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol 38: 980–983.R. YamamotoT. DemuraH. Fukuda1997Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells.Plant Cell Physiol38980983

4. Fukuda H (2004) Signals that control plant vascular cell differentiation. Nat Rev Mol Cell Biol 5: 379–391.H. Fukuda2004Signals that control plant vascular cell differentiation.Nat Rev Mol Cell Biol5379391

5. Neff MM, Nguyen SM, Malancharuvil EJ, Fujioka S, Noguchi T, et al. (1999) BAS1: a gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proc Natl Acad Sci U S A 96: 15316–15323.MM NeffSM NguyenEJ MalancharuvilS. FujiokaT. Noguchi1999BAS1: a gene regulating brassinosteroid levels and light responsiveness in Arabidopsis.Proc Natl Acad Sci U S A961531615323

6. Song L, Zhou XY, Li L, Xue LI, Yang X, et al. (2009) Genome-wide analysis revealed the complex regulatory network of brassinosteroid effects in photomorphogenesis. Mol Plant 2: 755–772.L. SongXY ZhouL. LiLI XueX. Yang2009Genome-wide analysis revealed the complex regulatory network of brassinosteroid effects in photomorphogenesis.Mol Plant2755772

7. Wada K, Marumo S, Ikekawa N, Morisaki M, Mori K (1981) Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol 22: 323–325.K. WadaS. MarumoN. IkekawaM. MorisakiK. Mori1981Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings.Plant Cell Physiol22323325

8. Wada K, Marumo S, Abe H, Morishita T, Nakamura K, et al. (1984) A rice lamina inclination test – a micro-quantitative bioassay for brassinosteroids. Agric Biol Chem 48: 719–726.K. WadaS. MarumoH. AbeT. MorishitaK. Nakamura1984A rice lamina inclination test – a micro-quantitative bioassay for brassinosteroids.Agric Biol Chem48719726

9. Sasse JM, Smith R, Hudson I (1995) Effect of 24-epibrassinolide on germination of seeds of Eucalyptus camaldulensis in saline conditions. Proc Plant Growth Regul Soc Am 22: 136–141.JM SasseR. SmithI. Hudson1995Effect of 24-epibrassinolide on germination of seeds of Eucalyptus camaldulensis in saline conditions.Proc Plant Growth Regul Soc Am22136141

10. Steber GM, McCourt P (2001) A role for brassinosteroids in germination in Arabidopsis. Plant Physiol 125: 763–769.GM SteberP. McCourt2001A role for brassinosteroids in germination in Arabidopsis.Plant Physiol125763769

11. Kim TW, Michniewicz MM, Bergmann DC, Wang ZY (2012) Brassinosteroid inhibits stomatal development by releasing GSK3-mediated inhibition of a MAP kinase pathway. Nature, Epub 2012/02/ 07: TW KimMM MichniewiczDC BergmannZY Wang2012Brassinosteroid inhibits stomatal development by releasing GSK3-mediated inhibition of a MAP kinase pathway.Nature, Epub 2012/02/07

12. Iwahori S, Tominaga S, Higuchi S (1990) Retardation of abscission of citrus leaf and fruitlet explants by brassinolide. Plant Growth Reg 9: 119–125.S. IwahoriS. TominagaS. Higuchi1990Retardation of abscission of citrus leaf and fruitlet explants by brassinolide.Plant Growth Reg9119125

13. Fujioka S, Yokota T (2003) Biosynthesis and metabolism of brassinosteroids. Annu Rev Plant Biol 54: 137–164.S. FujiokaT. Yokota2003Biosynthesis and metabolism of brassinosteroids.Annu Rev Plant Biol54137164

14. Li J, Nagpal P, Vitart V, McMorris TC, Chory J (1996) A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398–401.J. LiP. NagpalV. VitartTC McMorrisJ. Chory1996A role for brassinosteroids in light-dependent development of Arabidopsis.Science272398401

15. Szekeres M, Nemeth K, Koncz-Kalman Z, Mathur J, Kauschmann A, et al. (1996) Brassinosteriods rescue the deficiency of CYP90, a cytochrom P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171–182.M. SzekeresK. NemethZ. Koncz-KalmanJ. MathurA. Kauschmann1996Brassinosteriods rescue the deficiency of CYP90, a cytochrom P450, controlling cell elongation and de-etiolation in Arabidopsis.Cell85171182

16. Kauschmann A, Jessop A, Koncz C, Szekeres M, Willmitzer L, et al. (1996) Genetic evidence for an essential role of brassinosteroids in plant development. Plant J 9: 701–713.A. KauschmannA. JessopC. KonczM. SzekeresL. Willmitzer1996Genetic evidence for an essential role of brassinosteroids in plant development.Plant J9701713

17. Gachotte D, Meens R, Benveniste P (1995) An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast. Plant J 8: 407–416.D. GachotteR. MeensP. Benveniste1995An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast.Plant J8407416

18. Choe S, Noguchi T, Fujioka S, Takatsuto S, Tissier CP, et al. (1999) The Arabidopsis dwf7/ste1 mutant is defective in the a Δ7-sterol-C-5-desaturation step leading to brassinosteroid biosynthesis. Plant Cell 11: 207–222.S. ChoeT. NoguchiS. FujiokaS. TakatsutoCP Tissier1999The Arabidopsis dwf7/ste1 mutant is defective in the a Δ7-sterol-C-5-desaturation step leading to brassinosteroid biosynthesis.Plant Cell11207222

19. Choe S, Dilkes BP, Fujioka S, Takatsuto S, Sakurai A, et al. (1998) The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22-alpha-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10: 231–243.S. ChoeBP DilkesS. FujiokaS. TakatsutoA. Sakurai1998The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22-alpha-hydroxylation steps in brassinosteroid biosynthesis.Plant Cell10231243

20. Choe S, Dilkes BP, Gregory BD, Ross AS, Yuan H, et al. (1999) The Arabidopsis dwarf1 mutant Is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis. Plant Physiol 119: 897–907.S. ChoeBP DilkesBD GregoryAS RossH. Yuan1999The Arabidopsis dwarf1 mutant Is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis.Plant Physiol119897907

21. Clouse SD (2011) Brassinosteroids. The Arabidopsis Book 9: e0151.SD Clouse2011Brassinosteroids.The Arabidopsis Book9e0151

22. Fernandez MGS, Becraft PW, Yin Y, Lübberstedt T (2009) From dwarves to giants? Plant height manipulation for biomass yield. Trends in Plant Sci 14: 454–461.MGS FernandezPW BecraftY. YinT. Lübberstedt2009From dwarves to giants? Plant height manipulation for biomass yield.Trends in Plant Sci14454461

23. Hartwig T, Chuck GS, Fujioka S, Klempien A, Weizbauer R, et al. (2011) Brassinosteroid control of sex determination in maize. Proc Natl Acad Sci U S A 108: 19814–19819.T. HartwigGS ChuckS. FujiokaA. KlempienR. Weizbauer2011Brassinosteroid control of sex determination in maize.Proc Natl Acad Sci U S A1081981419819

24. Makarevitch I, Thompson A, Muehlbauer GJ, Springer NM (2012) Brd1 gene in maize encodes a brassinosteroid C-6 oxidase. PLoS ONE 7: e30798.I. MakarevitchA. ThompsonGJ MuehlbauerNM Springer2012Brd1 gene in maize encodes a brassinosteroid C-6 oxidase.PLoS ONE7e30798

25. Blackwell HE, Zhao Y (2003) Chemical genetic approaches to plant biology. Plant Physiol 133: 448–455.HE BlackwellY. Zhao2003Chemical genetic approaches to plant biology.Plant Physiol133448455

26. Raikhel N, Pirrung M (2005) Adding precision tools to the plant biologists' toolbox with chemical genomics. Plant Physiol 138: 563–564.N. RaikhelM. Pirrung2005Adding precision tools to the plant biologists' toolbox with chemical genomics.Plant Physiol138563564

27. Sekimata K, Han SY, Yoneyama K, Takeuchi Y, Yoshida S, et al. (2002) A specific and potent inhibitor of brassinosteroid biosynthesis possessing a dioxolane ring. J Agric Food Chem 50: 3486–3490.K. SekimataSY HanK. YoneyamaY. TakeuchiS. Yoshida2002A specific and potent inhibitor of brassinosteroid biosynthesis possessing a dioxolane ring.J Agric Food Chem5034863490

28. Rademacher W (1991) Biochemical effects of plant growth retardants. In: HW Gausman, editor. editor. Plant Biochemical Regulators. New York: Marcel Dekker. pp. 169–200.W. Rademacher1991Biochemical effects of plant growth retardants.Gausman HWeditorPlant Biochemical Regulators. New YorkMarcel Dekker169200

29. Mizutani M, Ohta D (2010) Diversification of P450 genes during land plant evolution. Annu Rev Plant Biol 61: 291–315.M. MizutaniD. Ohta2010Diversification of P450 genes during land plant evolution.Annu Rev Plant Biol61291315

30. Rademacher W (2000) Growth retardants: effects on gibberellin biosynthesis and other metabolic pathways. Annu Rev Plant Physiol Plant Mol Biol 51: 501–531.W. Rademacher2000Growth retardants: effects on gibberellin biosynthesis and other metabolic pathways.Annu Rev Plant Physiol Plant Mol Biol51501531

31. Yokota T, Nakamura N, Takahadhi N, Nonaka M, Sekimoto H, et al. (1991) Inconsistency between growth and endogenous levels of gibberellins, brassinosteroids, and sterols in Pisum sativum treated with uniconazole antipodes. In: Takahashi N, Phinney BO, Macmillan J, editors. Gibberellins. New York: Springer. pp. 339–349.T. YokotaN. NakamuraN. TakahadhiM. NonakaH. Sekimoto1991Inconsistency between growth and endogenous levels of gibberellins, brassinosteroids, and sterols in Pisum sativum treated with uniconazole antipodes.N. TakahashiBO PhinneyJ. MacmillanGibberellinsNew YorkSpringer339349

32. Iwasaki T, Shibaoka H (1991) Brassinosteroids act as regulators of tracheary-element differentiation in isolated Zinnia mesophyll cells. Plant Cell Physiol 32: 1007–1014.T. IwasakiH. Shibaoka1991Brassinosteroids act as regulators of tracheary-element differentiation in isolated Zinnia mesophyll cells.Plant Cell Physiol3210071014

33. Asami T, Yoshida S (1999) Brassinosteroid biosynthesis inhibitors. Trends Plant Sci 4: 348–353.T. AsamiS. Yoshida1999Brassinosteroid biosynthesis inhibitors.Trends Plant Sci4348353

34. Asami T, Min YK, Nagata N, Yamagishi K, Takatsuto S, et al. (2000) Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol 123: 93–100.T. AsamiYK MinN. NagataK. YamagishiS. Takatsuto2000Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor.Plant Physiol12393100

35. Asami T, Mizutani M, Fujioka S, Goda H, Min YK (2001) Selective interaction of triazole derivatives with dwf4, a cytochrome p450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta. J Biol Chem 276: 25687–25691.T. AsamiM. MizutaniS. FujiokaH. GodaYK Min2001Selective interaction of triazole derivatives with dwf4, a cytochrome p450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta.J Biol Chem2762568725691

36. Chung Y, Maharjan PM, Lee O, Fujioka S, Jang S, et al. (2011) Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis. Plant J 66: 564–578.Y. ChungPM MaharjanO. LeeS. FujiokaS. Jang2011Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis.Plant J66564578

37. Maharjan PM, Schulz B, Choe S (2011) BIN2/DWF12 antagonistically transduces brassinosteroid and auxin signals in the roots of Arabidopsis. J Plant Biol 54: 126–134.PM MaharjanB. SchulzS. Choe2011BIN2/DWF12 antagonistically transduces brassinosteroid and auxin signals in the roots of Arabidopsis.J Plant Biol54126134

38. Maharjan PM, Choe S (2011) High temperature stimulates DWARF4 (DWF4) expression to increase hypocotyl elongation in Arabidopsis. J Plant Biol 54: 425–429.PM MaharjanS. Choe2011High temperature stimulates DWARF4 (DWF4) expression to increase hypocotyl elongation in Arabidopsis.J Plant Biol54425429

39. Sekimata K, Kimura T, Kaneko I, Nakano T, Yoneyama K (2001) A specific brassinosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis, cress, tobacco, and rice. Planta 213: 716–21.K. SekimataT. KimuraI. KanekoT. NakanoK. Yoneyama2001A specific brassinosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis, cress, tobacco, and rice.Planta21371621

40. Gomes MMA (2011) Physiological effects related to brassinsteroid application in plants. In: Hayat S, Ahmad A, editors. Brassinosteroids: A Class of Plant Hormone. Dordrecht, The Netherlands: Springer Press. pp. 193–242.MMA Gomes2011Physiological effects related to brassinsteroid application in plants.S. HayatA. AhmadBrassinosteroids: A Class of Plant HormoneDordrecht, The NetherlandsSpringer Press193242

41. Vert G, Nemhauser JL, Geldner N, Hong F, Chory J (2005) Molecular mechanisms of steroid hormone signaling in plants. Annu Rev Cell Dev Biol 21: 177–201.G. VertJL NemhauserN. GeldnerF. HongJ. Chory2005Molecular mechanisms of steroid hormone signaling in plants.Annu Rev Cell Dev Biol21177201

42. Kim TW, Wang ZY (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu Rev Plant Biol 61: 681–704.TW KimZY Wang2010Brassinosteroid signal transduction from receptor kinases to transcription factors.Annu Rev Plant Biol61681704

43. Min YK, Asami T, Fujioka S, Murofushi N, Yamaguchi I, et al. (1999) New lead compounds for brassinosteroid biosynthesis inhibitors. Bioorg Med Chem Lett 9: 425–430.YK MinT. AsamiS. FujiokaN. MurofushiI. Yamaguchi1999New lead compounds for brassinosteroid biosynthesis inhibitors.Bioorg Med Chem Lett9425430

44. Yoshida Y, Aoyama Y (1991) Sterol I4a-demethylase and its inhibition: structural considerations on interaction of azole antifungal agents with lanosterol 14α-demethylase (P-45014DM) of yeast. Biochem Soc Trans 19: 778–782.Y. YoshidaY. Aoyama1991Sterol I4a-demethylase and its inhibition: structural considerations on interaction of azole antifungal agents with lanosterol 14α-demethylase (P-45014DM) of yeast.Biochem Soc Trans19778782

45. Wiggins TE, Baldwin BC (1984) Binding of azole fungicides related to dichlobutrazol to cytochrome P450. Pest Sci 14: 206–209.TE WigginsBC Baldwin1984Binding of azole fungicides related to dichlobutrazol to cytochrome P450.Pest Sci14206209

46. Li Z, Zlabek V, Velisek J, Grabic R, Machova J, et al. (2011) Multiple biomarkers responses in juvenile rainbow trout, Oncorhynchus mykiss, after acute exposure to a fungicide propiconazole. Environ Toxicol. Z. LiV. ZlabekJ. VelisekR. GrabicJ. Machova2011Multiple biomarkers responses in juvenile rainbow trout, Oncorhynchus mykiss, after acute exposure to a fungicide propiconazole.Environ ToxicolDOI:10.1002/tox.20701. DOI:10.1002/tox.20701.

47. Thorstenson CW, Lode O (2001) Laboratory degradation studies of bentazone, dichlorprop, MCPA, and propiconazole in Norwegian soils. J Environ Qual 30: 947–953.CW ThorstensonO. Lode2001Laboratory degradation studies of bentazone, dichlorprop, MCPA, and propiconazole in Norwegian soils.J Environ Qual30947953

48. Min YK, Asami T, Fujioka S, Murofushi N, Yamaguchi I, et al. (1999) New lead compounds for brassinosteroid biosynthesis inhibitors. Bioorg Med Chem Lett 9: 425–430.YK MinT. AsamiS. FujiokaN. MurofushiI. Yamaguchi1999New lead compounds for brassinosteroid biosynthesis inhibitors.Bioorg Med Chem Lett9425430

49. Wang ZY, Nakano T, Gendron J, He J, Chen M, et al. (2002) Nuclear-localized BZR1 mediates brassinosteroid-indiced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2: 505–513.ZY WangT. NakanoJ. GendronJ. HeM. Chen2002Nuclear-localized BZR1 mediates brassinosteroid-indiced growth and feedback suppression of brassinosteroid biosynthesis.Dev Cell2505513

50. Li L, Yu X, Thompson A, Guo M, Yoshida S, et al. (2009) Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression. Plant J 58: 275–286.L. LiX. YuA. ThompsonM. GuoS. Yoshida2009Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression.Plant J58275286

51. Belkhadir Y, Durbak A, Wierzba M, Schmitz RJ, Aguirre A, et al. (2010) Intragenic suppression of a trafficking-defective brassinosteroid receptor mutant in Arabidopsis. Genetics 185: 1283–1296.Y. BelkhadirA. DurbakM. WierzbaRJ SchmitzA. Aguirre2010Intragenic suppression of a trafficking-defective brassinosteroid receptor mutant in Arabidopsis.Genetics18512831296

52. Kim HB, Kwon M, Ryu H, Fujioka S, Takatsuto S (2006) The regulation of DWARF4 expression Is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis. Plant Physiol 140: 548–557.HB KimM. KwonH. RyuS. FujiokaS. Takatsuto2006The regulation of DWARF4 expression Is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis.Plant Physiol140548557

53. Clouse S, Feldmann K (1999) Molecular genetics of brassinosteroid action. In: Sakurai A, Yokota T, Clouse S, editors. Brassinosteroids: Steroidal Plant Hormones. Tokyo: Springer. pp. 163–190.S. ClouseK. Feldmann1999Molecular genetics of brassinosteroid action.A. SakuraiT. YokotaS. ClouseBrassinosteroids: Steroidal Plant HormonesTokyoSpringer163190

54. Liu K, Goodman M, Muse S, Smith JS, Buckler E, et al. (2003) Genetic structure and diversity among maize inbreed lines as inferred from DNA microsatellites. Genetics 165: 2117–2128.K. LiuM. GoodmanS. MuseJS SmithE. Buckler2003Genetic structure and diversity among maize inbreed lines as inferred from DNA microsatellites.Genetics16521172128

55. He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci U S A. 99: 10185–10190.JX HeJM GendronY. YangJ. LiZY Wang2002The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci U S A.991018510190

56. Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, et al. (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109: 181–191.Y. YinZY WangS. Mora-GarciaJ. LiS. Yoshida2002BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation.Cell109181191

57. Gampala SS, Kim TW, He JX, Tang W, Deng Z, et al. (2007) An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis. Dev Cell 13: 177–189.SS GampalaTW KimJX HeW. TangZ. Deng2007An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis.Dev Cell13177189

58. Bai MY, Zhang LY, Gampala SS, Zhu SW, Song WY, et al. (2007) Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice. Proc Natl Acad Sci U S A 104: 13839–13844.MY BaiLY ZhangSS GampalaSW ZhuWY Song2007Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice.Proc Natl Acad Sci U S A1041383913844

59. Tang W, Yuan M, Wang R, Yang Y, Wang C, et al. (2011) PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol 13: 124–131.W. TangM. YuanR. WangY. YangC. Wang2011PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1.Nat Cell Biol13124131

60. Noguchi T, Fujioka S, Takatsuto S, Sakurai A, Yoshida S, et al. (1999) Arabidopsis det2 Is defective in the conversion of (24R)-24-Methylcholest-4-En-3-One to (24R)-24-Methyl-5α-Cholestan-3-One in brassinosteroid biosynthesis. Plant Physiol 120: 833–840.T. NoguchiS. FujiokaS. TakatsutoA. SakuraiS. Yoshida1999Arabidopsis det2 Is defective in the conversion of (24R)-24-Methylcholest-4-En-3-One to (24R)-24-Methyl-5α-Cholestan-3-One in brassinosteroid biosynthesis.Plant Physiol120833840

61. Hedden P, Kamiya Y (1997) Gibberellin biosynthesis: enzymes, genes, and their regulation. Annu Rev Plant Physiol Plant Mol Biol 48: 431–460.P. HeddenY. Kamiya1997Gibberellin biosynthesis: enzymes, genes, and their regulation.Annu Rev Plant Physiol Plant Mol Biol48431460

62. Coles JP, Phillips AL, Croker SJ, García-Lepe R, Lewis MJ (1999) Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J 17: 547–556.JP ColesAL PhillipsSJ CrokerR. García-LepeMJ Lewis1999Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes.Plant J17547556

63. Xu Y, Gage DA, Zeevaart JAD (1997) Gibberellins and stem growth in Arabidopsis thaliana: effects of photoperiod on expression of the GA4 and GA5 loci. Plant Physiol 114: 1471–1476.Y. XuDA GageJAD Zeevaart1997Gibberellins and stem growth in Arabidopsis thaliana: effects of photoperiod on expression of the GA4 and GA5 loci.Plant Physiol11414711476

64. Xu YL, Li L, Gage DA, Zeevaart AD (1999) Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis. Plant Cell 11: 927–935.YL XuL. LiDA GageAD Zeevaart1999Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.Plant Cell11927935

65. Zimmerman P, Hirsch-Hoffman M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136: 2621–2632.P. ZimmermanM. Hirsch-HoffmanL. HennigW. Gruissem2004GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox.Plant Physiol13626212632

66. Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127: 1539–1555.J. DraperLAJ MurG. JenkinsGC Ghosh-BiswasP. Bablak2001Brachypodium distachyon. A new model system for functional genomics in grasses.Plant Physiol12715391555

67. Deleens E, Gregory N, Bourdu R (1984) Transition between seed reserve use and photosynthetic supply during development of maize seedlings. Plant Sci Lett 37: 35–39.E. DeleensN. GregoryR. Bourdu1984Transition between seed reserve use and photosynthetic supply during development of maize seedlings.Plant Sci Lett373539

68. Kim YS, Kim TW, Kim SK (2005) Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L. Phytochem 66: 1000–1006.YS KimTW KimSK Kim2005Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L. Phytochem6610001006

69. Owen WJ, Donzel B (1986) Oxidative degradation of chlortoluron, propiconazole, and metalaxyl in suspension cultures of various crop plants. Pest Biochem Physiol 26: 75–89.WJ OwenB. Donzel1986Oxidative degradation of chlortoluron, propiconazole, and metalaxyl in suspension cultures of various crop plants.Pest Biochem Physiol267589

70. Schnabel G, Dait Q, Paradkar MR (2003) Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola. Pest Manag Sci 59: 1143–1151.G. SchnabelQ. DaitMR Paradkar2003Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola.Pest Manag Sci5911431151

71. Schulz B, Kolukisaoglu HÜ (2006) Genomic analysis of plant ABC transporter genes. FEBS Lett 580: 1010–1016.B. SchulzHÜ Kolukisaoglu2006Genomic analysis of plant ABC transporter genes.FEBS Lett58010101016

72. Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophot Internat 11: 36–42.MD AbramoffPJ MagalhaesSJ Ram2004Image processing with ImageJ.Biophot Internat113642

73. Eggermont K, Goderis IJ, Broekaert WF (1996) High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glas beads. Plant Mol Biol Rep 14: 273–279.K. EggermontIJ GoderisWF Broekaert1996High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glas beads.Plant Mol Biol Rep14273279

74. Orlova I, Nagegowda DA, Kish CM, Gutensohn M, Maeda H, et al. (2009) The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta. Plant Cell 21: 4002–4017.I. OrlovaDA NagegowdaCM KishM. GutensohnH. Maeda2009The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta.Plant Cell2140024017

75. Zar JH (2010) Biostatistical analysis. Fifth edition. Prentice-Hall, Upper Saddle River, NJ. JH Zar2010Biostatistical analysis.Fifth edition. Prentice-Hall, Upper Saddle River, NJ