INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The application of many biologically active brassinosteroid (BR) homologs has been shown to cause remarkable growth responses in plants, including stem elongation, pollen tube growth, leaf bending, leaf unrolling, root inhibition, proton pump activation (Mandava, 1988), promotion of ethylene production (Schlagnhaufer and Arteca, 1991), tracheary element differentiation (Iwasaki and Shibaoka, 1991; Yamamoto et al., 1997), and cell elongation (Azpiroz et al., 1998). The functions of endogenous BRs have been revealed by identifying several BR-deficient Arabidopsis mutants, such as dwf1 (Choe et al., 1999a), cbb1 (Kauschmann et al., 1996), dwf4 (Choe et al., 1998), ste1/dwf7 (Gachotte et al., 1996; Choe et al., 1999b), cpd (Szekeres et al., 1996), and det2 (Li et al., 1996; Fujioka et al., 1997). Recently, dwarf mutants of pea (Nomura et al., 1997) and tomato (Bishop et al., 1999) have also been characterized as BR deficient. The above findings indicate that the use of BR-deficient mutants has been invaluable in investigating an essential role of BRs in plant growth and development, and, consequently, BRs have recently been recognized as a new class of phytohormones (Yokota, 1997; Clouse and Sasse, 1998).

The use of specific biosynthesis inhibitors is an alternative way for the determination of physiological functions of endogenous substances. As shown in mode-of-action studies on gibberellins (GAs), GA-deficient mutants and GA biosynthesis inhibitors are both quite effective (Rademacher, 1989; Kamiya and Hedden, 1997). Similarly, a specific inhibitor of BR biosynthesis can provide a new and complementary approach to understanding the functions of BRs (Yokota, 1999). KM-01 is the first reported selective BR inhibitor, but appears to be of limited use for probing the role of BRs in plants due to its very low activity when applied alone (Kim et al., 1995). Other than KM-01, there have been no BR inhibitors, but Yokota et al. (1991) observed a slight reduction in the concentration of endogenous castasterone when plants were treated with uniconazole, and Iwasaki and Shibaoka (1991) reported that this compound inhibited brassinolide-induced tracheary element differentiation. These observations imply that brassinolide biosynthesis is also affected, since uniconazole is known to block GA biosynthesis.

Various triazole compounds, including uniconazole, are known to inhibit many cytochrome P450s, a large and ubiquitous group of enzymes that catalyze oxidative processes in life systems (Rademacher, 1991), but inhibition of particular enzymes can be strictly controlled by specific inhibitors. This indicates that every enzyme has its own characteristic three-dimensional inhibitor binding site structure. Furthermore, many steps of BR biosynthesis are thought to be performed by cytochrome P450 enzymes, for example, conversion from campestanol to 6-hydroxycampestanol, 6-oxocampestanol to cathasterone, cathasterone to teasterone, typhasterol to castasterone, and castasterone to brassinolide (Sakurai and Fujioka, 1997). In this context, it would be beneficial to screen for a specific inhibitor of BR biosynthesis among triazole compounds. Eventually, we found some triazole derivatives to be good lead compounds for BR biosynthesis inhibitors (Min et al., 1999). Intensive study on structure-activity relationships of such lead compounds led us to the finding of a potent inhibitor, brassinazole, (Figure 1) (Asami and Yoshida, 1999). Brassinazole was synthesized on the basis of known methods (Buschmann et al., 1987), and is unique in that it has a tertiary hydroxy group on the carbon adjacent to the carbon where a triazole ring is attached, whereas other known triazolic PGRs have a secondary hydroxyl group at this position.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Structure of brassinazole. In this study, brassinazole was used as a racemic mixture.

We report the characterization of brassinazole as a BR biosynthesis inhibitor and examine the putative target sites of this chemical.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Arabidopsis mutants such as det2 and cpd show strong dwarfism with curly dark green leaves in the light and a de-etiolated phenotype with short hypocotyls and open cotyledons in the dark, which are characteristic of light-grown plants. This phenotype was rescued by the application of brassinolide, but the other plant hormones, such as auxin and GA, had no effect (Clouse and Sasse, 1998). Based on these facts, we tested brassinazole in the Arabidopsis seedling assay. Brassinazole markedly caused malformation of seedlings, which became morphologically similar to BR-deficient mutants (Fig. 2A). At a concentration higher than 1 µM, the phenotype became very similar to that of BR-deficient mutants. These brassinazole-induced phenotypes were rescued by co-application of 10 nM brassinolide (Fig. 2B). In the dark, brassinazole induced a de-etiolated phenotype with a short hypocotyl (Fig. 3A) and open cotyledons (Fig. 3B), similar to BR-deficient mutants. These phenotypes were rescued by the application of 10 nM brassinolide (Fig. 3A). Uniconazole, a potent GA biosynthesis inhibitor (Izumi et al., 1984), also induced short hypocotyls in Arabidopsis, which were returned to wild-type length by the application of 1 µM GA₃. However, brassinazole-induced short hypocotyls could not be rescued by GA₃ (Fig. 3C).

View larger version (29K): [in this window] [in a new window]   Figure 2.   Effect of brassinazole (Brz) on Arabidopsis seedlings grown in the light. A, Brassinazole (5, 1, and 0.5 µM)-treated Arabidopsis (14-d-old) show dwarfism in a concentration-dependent manner. B, Brassinazole (1 µM)-treated Arabidopsis (14-d-old) show a BR-deficient mutant-like phenotype, which is rescued by the application of brassinolide (BL) (10 nM). CONT, Control.

View larger version (34K): [in this window] [in a new window]   Figure 3.   Effect of brassinazole (Brz) on Arabidopsis seedlings grown in the dark. Brassinazole (1 µM)-treated Arabidopsis (7-d-old) shows BR-deficient mutant-like phenotype. A, Brassinazole-induced short hypocotyl and open cotyledon, which are rescued by brassinolide (BL) treatment (10 nM). B, Open cotyledon. C, Both uniconazole (U) (0.1 µM) and brassinazole (1 µM) induced short hypocotyl. GA3 (1 µM) treatment rescued uniconazole-induced short hypocotyl, but not the brassinazole-induced one. CONT, Control.

One of the common characteristics found in BR-biosynthesis-deficient mutants is a reduction in longitudinal growth, which could be due to either a reduced number of cells or a failure in cell elongation (Szekeres et al., 1996; Azpiroz et al., 1998; Choe et al., 1999). As shown in Figure 4, the dwarfism in brassinazole-treated Arabidopsis was due to the reduction in longitudinal growth. In contrast to non-treated Arabidopsis, hypocotyl cell length was reduced and the thickness of cell walls increased in the brassinazole-treated plants, whereas no differences were detected in the number of cells along the length of either organ between the brassinazole-treated and non-treated plants.

View larger version (136K): [in this window] [in a new window]   Figure 4.   Light microscopy of brassinazole-treated and non-treated Arabidopsis stems sectioned longitudinally and transversally. A, Cross-section of non-treated plant. B, Longitudinal section of non-treated plant. C, Cross-section of brassinazole-treated plant. Cell size is increased in different tissues. D, Longitudinal section of brassinazole-treated plant. Cell size is reduced drastically in many different tissues. Arabidopsis seedlings were grown for 10 d in the dark. Bar = 100 µm.

BR-deficient mutants accumulate light-regulated genes in the dark (Chory et al., 1991; Li et al., 1996; Szekeres et al., 1996). Hybridization of steady-state RNAs prepared from brassinazole-treated seedlings of Arabidopsis grown in the dark clearly showed that photomorphogenesis in brassinazole-treated Arabidopsis was accompanied by an increase in the expression of light-regulated genes coding for the small subunit of ribulose 1,5-diphosphate carboxylase (rbcS), chlorophyll a/b binding protein (cab), and the 32-kD QB-binding protein of photosystem II (psbA; Fig. 5). These levels were significantly higher than those in dark-grown non-treated seedlings, but in the light there was no difference between brassinazole-treated and non-treated seedlings. The dose-response test of brassinazole in the dark also demonstrated that the effect of brassinazole on reducing hypocotyl length became saturated at 3 µM or higher concentrations (Fig. 6).

View larger version (64K): [in this window] [in a new window]   Figure 5.   Accumulation of mRNAs for rbcS, cab, and psbA in light-grown and dark-grown brassinazole-treated and non-treated plants. Lane 1, Dark-grown non-treated plants; lane 2, dark-grown brassinazole-treated plants; lane 3, light-grown non-treated plants; lane 4, light- grown brassinazole-treated plants. Two micrograms of total RNA was loaded per lane. Bottom panel, Stained gel showing rRNAs.

View larger version (54K): [in this window] [in a new window]   Figure 6.   Dark-grown 10-d-old seedlings of Arabidopsis treated with different concentrations of brassinazole (Brz) from 0 to 10 µM. Hypocotyl length decreased as the concentration of brassinazole increased. The change in length occurred between 0.1 and 0.5 µM. Data are the means ± SE obtained from 20 seedlings. CONT, Control.

To examine whether brassinazole can cause a BR-deficient-mutant-like phenotype in plants other than Arabidopsis, brassinazole was also applied to cress, a plant that has been used previously to investigate the effects of brassinolide (Yopp et al., 1981; Jones-Held et al., 1996). The hypocotyl length of cress seedlings treated with brassinazole at 1 µM and higher was about 40% of the control (Fig. 7). This effect of 1 µM brassinazole on retarding the hypocotyl elongation of cress seedling was negated by the application of 10 nM brassinolide, but not by 1 µM GA. The hypocotyl length of cress seedlings treated with uniconazole at 1 µM or higher was also about 40% of the control, and this effect was negated by the application of GA but not brassinolide.

View larger version (36K): [in this window] [in a new window]   Figure 7.   Retardation of cress seedling growth by brassinazole (Brz) or uniconazole (U) and rescue by brassinolide or GA3. Brassinolide rescues plants treated with brassinazole but not uniconazole. GA3 added to plants rescues those treated with uniconazole but not brassinazole. CONT, Control.

Brassinazole is a triazole derivative, a number of which have been shown to inhibit cytochrome P450s. Therefore, brassinazole may also block a cytochrome P450, but specifically in the BR biosynthesis pathway. A BR biosynthesis pathway has been proposed that includes many steps likely to be catalyzed by a cytochrome P450 (Yokota, 1997). To investigate the biosynthetic step(s) affected by brassinazole, we examined the effect of biosynthetic intermediates downstream of cathasterone on hypocotyl elongation of both brassinazole-treated and non-treated etiolated Arabidopsis seedlings. As shown in Figure 8, 1 µM cathasterone and teasterone, 100 nM castasterone, and 10 nM brassinolide had no effect on non-treated seedlings. However, these concentrations of teasterone, castasterone, and brassinolide were effective in rescuing brassinazole-treated hypocotyl growth, respectively. Cathasterone had almost no effect on rescuing the brassinazole-treated hypocotyl growth. As in a similar experiment done by Fujioka et al. (1997), both cathasterone and teasterone rescued the defective hypocotyl growth of the dark-grown det2 mutant. Our results suggest that at least one of the target sites of brassinazole in BR biosynthesis inhibition is the oxidation step of cathasterone to teasterone, which is catalyzed by CPD (Szekeres et al., 1996), a P450 cytochrome. Therefore, brassinazole exhibits its effect by reducing the supply of brassinolide in the plant. In the reversion test, plants tended to be sensitive to growth conditions, maybe because of slow uptake and transport of brassinolide within cress and/or a non-specific effect(s) of brassinazole on other aspects of plant metabolism. This could be why brassinolide did not completely reverse the inhibition by brassinazole, as shown in Figure 8.

View larger version (52K): [in this window] [in a new window]   Figure 8.   Brassinazole (Brz)-treated Arabidopsis hypocotyl elongation in the dark in response to applied cathasterone (CT), teasterone (TE), castasterone (CS), and brassinolide (BL). Data are the means ± SE obtained from 30 seedlings. CONT, Control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Both in the dark and in the light, brassinazole-induced morphological changes were nullified by the addition of brassinolide. In the dark, brassinazole-treated Arabidopsis develop as light-grown plants and express light-regulated genes, as do BR-deficient mutants. Light microscopic analysis of hypocotyls indicated that the brassinazole-induced dwarfism was due to a failure of cell elongation, as was also observed in the BR-deficient mutants cpd, dwf4, and dwf7. The cpd and det2 mutations induce de-etiolation and expression of light-induced genes in the dark, which were also seen in brassinazole-treated Arabidopsis. These results strongly suggest that brassinazole treatment caused brassinolide deficiency in Arabidopsis.

Feeding experiments demonstrated that brassinazole-induced retardation of hypocotyl growth was rescued by the addition of exogenous BR biosynthesis intermediates such as teasterone, but not by cathasterone, suggesting that the inhibition site of brassinazole may be the step from cathasterone to teasterone. The enzyme involved in this step mediates a hydroxylation at C-23 of the side chain of cathasterone and is known to be encoded by CPD, which has been proposed to be a novel cytochrome P450 (Szekeres et al., 1996). Considering that brassinazole is in a chemical class of triazole derivatives that exhibit their activities by inhibiting cytochrome P450s, it is likely that brassinazole activity is caused by this enzyme and possibly others as well. In some cases, triazole derivatives have multiple inhibition sites. For example, GA biosynthesis inhibitors blocking ent-kaurene oxidation can also affect other cytochrome P450 monooxygenases, although in most cases at a far lower degree of activity; for example, in the inhibition of sterol formation by the blocking of 14-demethylation and the inhibition of the oxidative inactivation of abscisic acid (Rademacher, 1991). Therefore, secondary effects of brassinazole may also play a role in plants, but subsequent results indicated that brassinazole showed little effect on GA biosynthesis at 1 µM or lower concentrations. That is, uniconazole disrupts Arabidopsis germination due to the inhibition of GA biosynthesis, while brassinazole has no effect on germination. Morphological changes induced by brassinazole are rescued by the addition of exogenous brassinolide but not by GA.

The C-22 and C-23 positions of BRs are successively hydroxylated by cytochrome P450s encoded by DWF4 and CPD, respectively. The enzymes catalyzing these two steps are different; however, not only are their functions similar but the DNA sequences are similar to each other (Choe et al., 1998). Taking the similar function of the enzyme active sites into consideration, it is possible to speculate that brassinazole inhibits both steps. But in this case, brassinazole-treated Arabidopsis does not show a response to cathasterone or to earlier intermediates of BR biosynthesis such as campestanol and 6-oxocampestanol. This low response makes it difficult to investigate the target sites of brassinazole upstream of cathasterone by feeding experiments. In BR biosynthesis, Fujioka and Sakurai (1997) have demonstrated that there are at least two branched biochemical pathways to the end product brassinolide: the early and the late C-6 oxidation pathways (Fujioka and Sakurai, 1997). In the late C-6 oxidation pathway, there are two hydroxylation steps of the side chains of campestanol to 6-deoxoteasterone via 6-deoxocathasterone. These steps in the late C-6 oxidation pathway are very similar to those in the early C-6 oxidation pathway. On the basis of these experiments, we cannot rule out that brassinazole may attack these sites as well as the step from cathasterone to teasterone. Further investigations will answer these questions.

In this report we demonstrate that brassinazole induces morphological changes in plants by interfering with the biosynthesis of BRs. At present, BR-deficient mutants are known only in Arabidopsis, tomato, and pea. This novel BR biosynthesis inhibitor will play an important role in investigations into the function of BRs not only in other plants, but also in tissues, organs, and biochemical processes. As shown in Figure 6, we varied the concentration of BRs in plants by varying the concentration of brassinazole. This may make it possible to titrate the minimum concentration of BRs for the growth of plants by comparing the concentration of BRs in brassinazole-treated and non-treated plants. Moreover, the ability to select a group of new mutants using GA biosynthesis inhibitors (Jacobsen and Olszewski, 1993; Nambara et al., 1994) suggests that this inhibitor will provide a way to find a new BR pathway or other novel mutants. Other than its use in basic science, brassinazole can be developed as a new commercial plant growth regulator.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Chemicals

Brassinolide and castasterone were purchased from CIDtech Research (Cambridge, Ontario, Canada). Other intermediates in the BR biosynthesis pathway used in this report were synthesized as described previously (Takatsuto et al., 1984; Takatsuto, 1986; Fujioka et al., 1995). Murashige and Skoog salt and vitamin mixture was purchased from Gibco-BRL (Grand Island, NY). Brassinazole was synthesized on the basis of known methods (Buschmann et al., 1987). Brassinazole exists as enantiomers, and the structure-activity relationships of brassinazole and other triazole derivatives will be reported elsewhere.

Plant Materials and Growth Conditions

Wild-type seeds of Arabidopsis (ecotype Columbia) were purchased from LEHLE Seeds (Round Rock, TX). Cress (Lepidium sativum) seeds were purchased locally. Wild-type and det2 seeds were cold treated (4°C) for 2 d, then surface-sterilized in 1% (w/v) solution of NaOCl for 20 min and washed with sterile distilled water five times. Seeds were sown on 1% (w/v) agar-solidified medium containing 0.5× Murashige and Skoog salts and 1.5% (w/v) Suc in plastic plates with or without chemicals. Wild-type and det2 plants were grown in 16-h light (240 µE m² s¹) and 8-h dark conditions in a growth chamber (22°C). The plates were sealed with Parafilm (American National Can, Chicago) for the screening experiment. For the rescue experiment, which required a longer experimental period, seeds were sown on 1% (w/v) agar-solidified medium containing 0.5× Murashige and Skoog salts and 1.5% (w/v) Suc in Agripots (Kirin Brew., Tokyo). Plants were grown in 16-h light (240 µE m² s¹) and 8-h dark conditions in a growth chamber (25°C). Cress seeds were sown on 1% (w/v) agar-solidified medium containing 0.5× Murashige and Skoog salts and 1.5% (w/v) Suc in Agripots with or without chemicals. Plants were grown in 16-h light (240 µE m² s¹) and 8-h dark conditions in a growth chamber (25°C).

The samples were fixed in 4% (w/v) paraformaldehyde buffered with 20 mM sodium cacodylate at pH 7.0 for 20 h at 4°C, dehydrated through an ethanol series, and then embedded in resin (Technovit 7100, Kulzer and Co., Wehrheim, Germany). The sections (2 µm thick) were cut with a glass knife on an ultramicrotome (ULTRACUT UCT, Leica, Wien, Austria), placed on cover slips, and dried. They were stained with 0.5% (w/v) toluidine blue O in 0.1 M phosphate-buffered saline (pH 7.0) for 30 s, and then washed in distilled water for 10 s. The samples were observed with a microscope (model IX70, Olympus, Tokyo).

RNA filter hybridizations were performed using standard molecular techniques (Sambrook et al., 1989) with slight modifications. For hybridization of RNA blots, the cDNA probes used were the same as described previously (Deng et al., 1991).