Rice BRD2: 24-methylenecampesterol to campesterol, reaction 2
DWARF1 and LKB mediate the C-24 reduction reactions in the early steps of BR biosynthesis in Arabidopsis and pea, respectively (Choe et al. 1999, Nomura et al. 1999). A double bond at C-24 is reduced to a single bond via two consecutive steps consisting of isomerization of Δ24 to Δ22 and subsequent reduction of the C-22 double bond (Choe et al. 1999, Nomura et al. 1999). Recently, Hong et al. (2005) reported that rice brassinosteroid-deficient dwarf2 (brd2) is defective in the rice gene that is homologous to Arabidopsis DWARF1. In the brd2 mutant plants, the endogenous amount of 24-methylenecholesterol, which is a precursor of the C-24 reduction step, accumulated more than 10-fold, whereas amount of the product, campesterol, was 1/1000th compared with wild-type. Interestingly, Hong et al. (2005) also noticed that the overall phenotypes of rice brd2 mutant were milder than those of rice brd1 mutant, which is defective in the BR C6-oxidation step. After examination of the endogenous BR levels, Hong et al. found that dolichosterone (DS) and other C-24 non-reduced BRs are enriched in brd2. Thus, they postulated that the C-24 reduction step could be skipped during BR biosynthesis. After careful bioassay of the rice lamina-bending system, they found that DS is also bioactive and induces typical bending at lamina joints. The accumulation of DS may explain the relatively weaker phenotypes of the brd2 mutants.
CYP90D2: 6-deoxoTE/TE to 3-dehydro-6-deoxoTE/3-dehydroTE (‘/’ separates the intermediates of the late and early C-6 oxidation pathways), reaction 10
Hong et al. (2003) reported that rice BR dwarf mutants including ebisu dwarf (d2) display characteristic phenotypes, such as inhibition of elongation in the second internode, erect leaves, and de-etiolation in the dark. On the basis of successful complementation of the mutant phenotype by exogenous application of BL, it was confirmed that d2 was defective in BL biosynthesis. A map-based cloning approach revealed that D2 encodes a novel member of a cytochrome P450 enzyme, CYP90D2 (Hong et al. 2003). Through a search of the rice genome databases, the authors found another close homolog that shares 66% identity in amino acid sequence level with D2, named CYP90D3. Currently, the CYP90D group consists in three members, two from rice and one from Arabidopsis (CYP90D1, Nelson et al. 2004b). Recently, Kim et al. (2005) proposed that Arabidopsis CYP90D1 is involved in the BR 3-dehydrogenation steps such as 6-deoxoTE/TE to 3-dehydro-6-deoxoTE/3-dehydroTE, based on the results of genetics and endogenous BR level analysis.
To elucidate a specific enzymatic step mediated by CYP90D2 and D3 of rice, Hong et al. (2003) performed intermediate feeding tests. The rice lamina-bending assay showed that only the biosynthetic intermediates 3-dehydroTE (3-dehydro-6-deoxo-TE) and its downstream compounds could induce lamina bending, whereas application of the upstream compounds such as TE and 6-deoxoTE resulted in negligible responses. Furthermore, analysis of endogenous levels of BRs showed that the level of 6-deoxoCT slightly increased, while the 3-dehydro-6-deoxo-TE level was only half that of wild-type. On the basis of the results of endogenous BR levels and feeding tests, it has been proposed that CYP90D2 mediates the 3-dehydrogenase reaction in the BR biosynthetic pathways.
CYP724B1 (DWARF11): 3-dehydro-6-deoxoTE/3-dehydroTE to 6-deoxoTY/TY, reaction 11
Similar to the reduced seed length phenotypes of Arabidopsis BR dwarfs (Choe et al. 2000), rice BR-defective mutants often display a short seed phenotype (Tanabe et al. 2005). To elucidate the underlying mechanisms of this short seed phenotype, Tanabe et al. took a map-based cloning approach to isolate the responsible gene for the dwarf11 (d11) mutant. The amino acid sequence of the cloned gene revealed that it shares 43% identity with Arabidopsis DWF4 (CYP90B1), suggesting a role in BR biosynthesis. To identify the possible enzymatic step in the pathways that this gene product participated in, these authors used the rice lamina-bending assay with various biosynthetic intermediates. In the BR biosynthetic pathways, only the intermediates from 6-deoxoTY and its downstream compounds could induce bending responses from the both d11-1 and d11-2 mutant plants (Tanabe et al. 2005). This suggests that d11 is defective in a step of BR biosynthetic pathways that converts 6-deoxo-3-dehydroTE/3-dehydroTE to 6-deoxo-TY/TY.
Arabidopsis CYP85A2 and tomato CYP85A3: 6-deoxoCS to BL via CS, reaction 13
BL is an end product in the BR biosynthetic pathways and is considered to be the most active compound among approximately 50 BRs discovered to date. Thus, identification of the enzyme that mediates the final step in the BL biosynthetic pathways has been a focal point in this research. Two different groups independently identified this enzyme, which is a Baeyer-Villiger type oxidase. The Yamaguchi group at RIKEN (Nomura et al. 2005) reported that a homolog (CYP85A3) of tomato Dwarf enzyme (CYP85A1) mediates BR-6-hydroxylation as well as the Baeyer-Villiger type oxidation step. The tomato Dwarf gene was initially discovered by Bishop et al. (1996). A transposon-tagged loss-of-function mutant, extreme dwarf (dx), displayed severe dwarfism due to a block in the BR-6-oxidation step, which is the penultimate step in the pathways (Bishop et al. 1996). Cloning of the gene responsible for the dx mutant revealed that a Cytochrome P450 (CYP85A1) is disrupted.
Unexpectedly, Nomura et al. (2005) found that dx fruit contained significant amounts of BL. Because BL is synthesized using CS as a precursor, they hypothesized that an alternative enzyme substituting for CYP85A1 may exist in the dx fruit. To clone this second copy of the CYP85A1 gene active in tomato fruit, they designed a pair of oligonucleotides based on the nucleotide sequence identity conserved in the CYP85 family of genes already known in Arabidopsis, tomato, and pea. PCR amplification of a DNA fragment using the designed oligonucleotides led to the isolation of a gene that shares 75% identity in amino acid sequence level with CYP85A1, which was named CYP85A3. As expected, the spatial expression pattern of the two CYP85 genes differed in tomato tissues; CYP85A3 was preferentially expressed in the fruits whereas CYP85A1 expressed in vegetative tissues. Thus, it was hypothesized that the elevated level of BL in the dx fruit was due to the enzymatic activity of CYP85A3. To test whether CYP85A3 indeed mediates the BR-6-oxidation reaction, CYP85A3 was heterologously expressed in yeast, and feeding experiments were performed. As was the case for CYP85A1 (Bishop et al. 1999), feeding of a deuterium- labeled substrate, [2H6]6-deoxoCS, to the CYP85A3-expressing yeast strain resulted in the production of [2H6]6-CS (Nomura et al. 2005). Surprisingly, further examination of the GC-MS profiles of the reaction products from [2H6]6-deoxoCS feeding experiments showed that in addition to the expected [2H6]6-CS peak, the authors could identify another peak that was similar to [2H6]6-BL. Comparison of the characteristic ions, m/z, from standard BL to that of the metabolic profile confirmed that the peak observed in CY85A3 was the same as BL. This means that CYP85A3 is a multifunctional enzyme that mediates three consecutive enzymatic steps in the BL biosynthetic pathways, namely C-6 hydroxylation, dehydrogenation, and Baeyer-Villiger type oxidation.
Independently, Kim et al. (2005) also found that Arabidopsis CYP85A2 mediates the final step in the BL biosynthetic pathways. Previously, they found that tomato has a significant pool of C27-BRs including 28-norCS, and these C27-BRs are bioactive in various bioassays (Kim et al. 2004). Similarly to the experiments with tomato, it was shown that the Arabidopsis enzyme preparation could convert 28-norCS to CS, suggesting that Arabidopsis has a pathway from cholesterol to BL in addition to the conventional CR to BL pathway (Kim et al. 2004). On the basis of these findings, Kim et al. (2005) hypothesized that CYP85A1 and CYP85A2 from Arabidopsis have different substrate preferences: one for deoxoCS and the other for 28-norCS. To test this hypothesis, they heterologously expressed the two genes in yeast, and performed a metabolite conversion analysis. Results proved that CYP85A2 has significantly greater overall enzymatic activity than CYP85A1, and that CYP85A2 can effectively transform more of 6-deoxo-28-norCS to 28-norCS relative to CYP85A1 (Kim et al. 2005). In addition to the differential enzyme activity, Kim et al. found that CYP85A2-expressing yeast produce a BL-like peak when they feed the cell line with either of the two precursors, 6-deoxo-28-norCS or deoxoCS. Further comparison of the BL-like peak with the profile of authentic BL revealed that the peak was indeed BL that was produced after conversion from the precursors.
There has been debate over whether the nascent BR biosynthetic intermediates are bioactive, i.e. whether intermediates need be converted to BL to get bioactivity, or whether some of BRs such as CS and TY possess nascent bioactivity without being metabolized to BL. Previously, Wang et al. (2001) showed that the BR receptor BRI1 can bind to both CS and BL, suggesting that CS might serve as a bioactive compound. In addition, thorough analysis of endogenous BR levels in different plants showed that rice and pea lack BL (Fujioka and Yokota 2003). Therefore, it has been suggested that both CS and BL are bioactive compounds. Genetic analysis of Arabidopsis mutants that are defective in either CYP85A1 or CYP85A2 gene revealed important findings concerning the nascent bioactivity of CS or BL. When the function of the CYP85A1 gene was disrupted, the mutant did not display any noticeable defects in development (Kim et al. 2005, Kwon et al. 2005, Nomura et al. 2005), possibly due to functional complementation by a redundant gene CYP85A2. However, a loss-of-function mutant for the CYP85A2 gene displayed semi-dwarf phenotype (Kim et al. 2005). Compared with a wild-type plant, rosette leaves look darker-green, rounder, and curled. In addition, filament growth was not sufficient to reach the stigmatic papillae, causing reduced fertility in the mutant (Kim et al. 2005). This suggests that BL deficiency in the cyp85a2 mutant negatively affected the reproductive organ development.
In contrast to the semi-dwarf phenotype of the cyp85a2 mutant, a double mutant defective in the two genes, cyp85a1 and cyp85a2, displayed the conventional BR dwarf phenotype (Kwon et al. 2005, Nomura et al. 2005). The double mutants possessed reduced stature, short petioles and pedicels, and dark green and rounder shape of rosette leaves (Kwon et al. 2005). In addition, the double mutant displayed a shorter hypocotyl length in the dark (Kwon et al. 2005), a characteristic of de-etiolation phenotypes of BR dwarf mutants (Choe 2004). Interestingly, the short hypocotyl in the dark was further reduced in presence of the BR biosynthetic inhibitor brassinazole. In addition, Kwon et al. (2005) also found that the dwarf phenotypes observed in the double mutants were relatively weaker than the cases of dwf7-1 or cpd-388, suggesting that the double mutant may maintain some minor pool of bioactive BRs. Examination of the endogenous BRs in the double mutants revealed that the amount of 6-deoxoCS was quadrupled, whereas the amount of CS was only 1/30th that of wild-type, possibly due to the block in the BR-6-oxidation step. However, the amounts of CN and 6-oxoCN in the double mutant were not significantly different from those of wild-type, suggesting that the BR-6-oxidation of CN (reaction 7) might be mediated by an enzyme other than the two CYP85s in Arabidopsis (Kwon et al. 2005).
The relatively mild growth defects observed in the Arabidopsis cyp85a2 mutant that is defective in the BL synthase suggests that BL might play a specific role. The developmental anomalies in a single mutant of CYP85A2 were most obvious in the reproductive organs (Kim et al. 2005). Similarly, tomato CYP85A3 expression is greatly enriched in reproductive organs and fruits (Nomura et al. 2005). In addition, Shimada et al. (2003) reported that Arabidopsis floral clusters and seeds accumulate significant amounts of BL relative to other organs, such as stems and leaves. Enrichment of BL in reproductive organs and relatively weak overall growth defects in the cyp85a2 single mutant of Arabidopsis suggest that CS is a bioactive compound responsible for overall vegetative growth, whereas BL could be more responsible for the development of reproductive organs. However, it cannot be ruled out that BL plays a role in the development of other vegetative organs. Cell-specific localization of BL and more thorough analysis of the BL synthase gene expression may shed light on the specific roles assigned to BL.
As mentioned earlier, BL has never been detected from rice plants. Thus, it is possible that rice lacks the homolog of BL synthase, CYP85A2. Currently, searching the rice pseudomolecules database (http://www.tigr.org) deduced from rice genome using Arabidopsis CYP85A2 as a probe revealed only one member of the CYP85 family. Heterologous expression of this gene and subsequent characterization should reveal whether this rice protein mediates only BR-6-oxidation.