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Castasterone is a likely end product of brassinosteroid biosynthetic pathway in rice
Biochemical and Biophysical Research Communications 374 (2008) 614–619
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Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c
Castasterone is a likely end product of brassinosteroid biosynthetic pathway in rice Bo Kyung Kim a, Shozo Fujioka b, Suguru Takatsuto c, Masafumi Tsujimoto b, Sunghwa Choe a,* a
Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea Laboratory of Cellular Biochemistry, RIKEN, Wako-shi, Saitama 351-0198, Japan c Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan b
a r t i c l e
i n f o
Article history: Received 9 July 2008 Available online 24 July 2008 Keywords: Brassinolide synthase Brassinosteroids CYP85 Cytochrome P450 Phytosteroids Steroid hormones Arabidopsis Rice
a b s t r a c t Brassinolide is known to be the most biologically active compound among more than 50 brassinosteroids identified to date. However, brassinolide has not been detected in rice. To determine if this is due to the lack of the brassinolide synthase function in the rice CYP85A enzyme, we performed analyses to study metabolic conversion using a yeast strain harboring the rice CYP85A1 gene. In repeated feeding tests where the substrates were used, the biosynthetic pathway progressed only up to the synthesis of castas terone, not of brassinolide. Phylogenetic analysis of the CYP85 amino acid sequences revealed that dupli cation of the CYP85 gene has occurred in most dicotyledonous plant genomes; further, 1 of the 2 copies of CYP85 is evolving to develop a brassinolide synthase function. However, only a single copy of this gene is found in the currently available genome sequences of graminaceous plants; this is a likely explanation for the absence of an endogenous pool of brassinolide in rice plants. © 2008 Elsevier Inc. All rights reserved.
Brassinosteroids (BRs) are a group of natural compounds that possess a steroid backbone with oxidized carbon atoms at multiple sites in the core ring and the side chain (Fig. 1). To date, more than 50 BR compounds have been identified from 61 plants across the plant kingdom [1]. Bioassays such as rice lamina inclination assay [2–4] proved brassinolide (BL), the most highly oxidized form among BRs, the most biologically active. Campesterol (CR) is the precursor of the BL biosynthetic pathway; this pathway is chan neled through 2 alternative routes depending on the order of 2 reactions, namely, C-22 hydroxylation and C-5 reduction. The C-22 hydroxylation reactions are mediated by the Arabidopsis thaliana DWARF4 protein, which belongs to the cytochrome P450 monooxy genase family CYP90B1 [5,6]. The resulting C-22 hydroxylated com pounds are then hydroxylated at the C-23 position by CYP90C1 and CYP90D1 [7] before being subjected to reduction at position C-5. Thereafter, these compounds undergo C-3 epimerization and C-2 hydroxylation reactions, and finally 3 consecutive C-6 oxidation reactions occur, leading to the generation of castasterone (CS) and BL [8]. BL is mainly derived from 6-deoxocastasterone (6-deoxoCS) by the catalytic actions of a group of cytochrome P450 enzymes belonging to the CYP85 family; these enzymes include Arabidopsis CYP85A2 (AtCYP85A2) and Lycopersicon esculentum CYP85A3
* Corresponding author. Fax: +82 2 872 1993. E-mail address: shc[email protected] (S. Choe). 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.07.073
(LeCYP85A3). These enzymes mediate 3 consecutive steps in the BL biosynthetic pathway: C-6 hydroxylation, C-6 dehydrogenation, and a Baeyer–Villiger-type oxidation to create a lactone at ring B of the steroid backbone (Fig. 1) [9,10]. The Arabidopsis genome possesses 2 copies of the CYP85 gene; due to the functional redun dancy between these 2 genes, plants exhibit conventional BR dwarf phenotype only when the functions of both the genes are simulta neously disrupted [9,10]. In contrast to the Arabidopsis and tomato genomes, the com pleted sequence of the rice genome revealed only 1 copy of the CYP85 gene. In addition, BL is not detectable even in Osbri1 mutant plants, which are assumed to accumulate large amounts of BRs in vivo [11,12]. To examine if the rice CYP85A1 (OsCYP85A1) enzyme functions as a CS synthase rather than both CS synthase and BL syn thase, we expressed the OsCYP85A1 gene in yeast and performed analyses to study metabolic conversion. B a eye r-V i l l i ge r- t y p e
Materials and methods Cloning and heterologous expression of AtCYP85A2 and OsCYP85A1. The cDNAs encoding AtCYP85A2 and OsCYP85A1 enzymes were cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the mRNA isolated from Arabidopsis and rice seedlings. The oligonucleotide sequences are as follows: OsCYP85-F, 59-CACCATGGTGTTGGTGGC-39; OsCYP85R, 59-GCAGTAATCTTGAACGCGGATAT-39; AtCYP85A2-F, 59-CACC ATGATGATGATTTTGGGT-39; and AtCYP85A2-R, 59-GCAGTAAG t ra n s c r i pta s e – p o ly m e ra s e
5 9 - CAC CATGATGATGAT T T TG G GT- 3 9 ;
5 9 - G CAGTA AG GTGA ACAC T TA AG - 3 9 .
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
615
OH OH OH OH
HO
1 AtCYP85A1 AtCYP85A2 LeCYP85A1 LeCYP85A3 PsCYP85A1 PsCYP85A6 OsCYP85A1
H
6-Deoxotyphasterol (6-DeoxoTY) PsCYP92A6
HO
H
O
Typhasterol (TY) OH
OH OH OH
HO
1
HO
HO HO
H
H
6-Deoxocastasterone (6-DeoxoCS)
Castasterone (CS)
O
AtCYP85A2 LeCYP85A3
OH
2
OH
HO
HO H
O
Brassinolide (BL)
O
Fig. 1. Part of the brassinolide biosynthetic pathway. The pathway precursor, campesterol, is converted to 6-deoxoTY via a series of reactions mediated by CYP90 enzymes and steroid 3-dehydrogenases. The carbon at the C-6 position of 6-deoxoTY and 6-deoxoCS is oxidized by CYP85 proteins to yield TY and CS, respectively (Reaction 1). Finally, CS is further oxidized to BL by the BL synthase action of CYP85 enzymes such as Arabidopsis CYP85A2 and tomato CYP85A3 (Reaction 2).
GTGAACACTTAAG-39. The cDNA fragments were subcloned into a pYES_DEST52 yeast expression vector (Invitrogen, USA) containing a uracil (URA) selection marker according to the protocol provided by the manufacturer. The cloned vector was transformed into the WAT11 strain for optimized expression of plant P450 proteins [13]. The selected transformants were subcultured and their respiratory capability was tested by observing their growth on glycerol-con taining N3 medium according to the previously described proto cols [14]. Site-specific mutagenesis was performed by PCR using overlap ping nucleotides containing the designated mutations: FOR, 59-tc gccgcctggtcctcctccgccgtcgtcgacatccaggc-39; and REV, 59-cctggatgt cgacgacggcggaggaggaccaggcggcga-39. Deletion of the 3 target Ser residues was confirmed by DNA sequencing using the M13 primer located in the expression vector. Quantitative analysis of sterols and BRs using gas chromatogra phy-mass spectrometry. The endogenous levels of both sterols and BRs were determined using 4-month-old rice plants according to the methods described previously [14]. In case of Arabidopsis, the levels in ‘‘Wassilewskija-2 (Ws-2)” ecotype was similarly deter mined using the aerial parts of 5-week-old adult plants grown in soil under regular long day conditions (16:8 h, light:dark). Protein gel-blot analysis. To check whether the transgene was correctly expressed, the total proteins were isolated from the yeast cells, and 20 lg of each protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Pro teins resolved by SDS–PAGE were transferred to nitrocellulose membranes (Amersham Bioscience, USA), and the protein bands were visualized by immunoblotting followed by incubation with anti-V5 antibodies (Invitrogen, USA) conjugated to horseradish per oxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnol ogy, USA). i n ‘ ‘ Wa s s i l ews k ij a - 2 ( W s - 2 ) ’ ’
( 16 h : 8
l i g h t: d a rk ) .
s u l fa te - p o lya c r yl a m i d e
Multiple sequence alignment and phylogenetic analysis. To study the distribution of CYP85 genes in plants, the GenBank database was searched using the rice CYP85A1 amino acid sequence as a probe. Sequences sharing greater than 55% identity were selected for fur ther comparison. Since full-length cDNA sequences for maize and Brachypodium were not available, virtual cDNA sequences were constructed using the Brachypodium shotgun sequences released by the Joint Genome Institute (JGI) covering the Brachypodium genome to a depth of 4£ and expressed sequence tags (ESTs) before obtaining the deduced amino acid sequences. The data base accession numbers for the CYP85 protein sequences are as follows: NP001050623 (rice), ABH01181 (barley), BAF56235 (pea; CYP85A1), BAF56236 (pea; CYP85A6), ABB60086 (grape; CYP85A1), CAO41843 (grape; CYP85A6), Q43147 (tomato; CYP85A1), Q50LE0 (tomato; CYP85A3), Q940V4 (Arabidopsis; CYP85A2), and Q9FMA5 (Arabidopsis; CYP85A1). Sequences for Populus-CYP85A1 and Popu lus-CYP85A4 were obtained from the cytochrome P450 database (http://drnelson.utmem.edu/CytochromeP450.html). A phylogenetic tree was constructed based on the full-length deduced amino acid sequences by using the neighbor-joining method of the CLUSTALW program (Version 1.83). Bootstrap mode (1000 replications) was used to estimate the confidence level that can be assigned to the branching nodes in the tree. full length
( 1, 0 0 0
Results and discussion
( S D S - PAG E )
S D S - PAG E
The endogenous level of BL is not detectable in rice Table 1 summarizes the endogenous BR levels in rice and Arabid opsis; the data presented in this table indicate that all the sterols that serve as precursors of BR biosynthesis were detected in the study. However, 2 BRs—CT and BL—were not produced at a detect
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Table 1 Endogenous levels of sterols and BRs in the rice cultivar “Dongjin” and Arabidopsis “Wassilewskija-2” wild-type Rice
Arabidopsis
Sterols 24-Methylenecholesterol (24-MC) Campesterol (CR) Campestanol (CN) 6-Oxocampestanol (6-OxoCN)
2490 35,100 823 10.2
5340 35,300 978 31.6
BRs 6-Deoxocathasterone (6-DeoxoCT) 6-Deoxoteasterone (6-DeoxoTE) 6-Deoxotyphasterol (6-DeoxoTY) 6-Deoxocastasterone (6-DeoxoCS) Cathasterone (CT) Typhasterol (TY) Castasterone (CS) Brassinolide (BL)
0.42 0.04 2.9 0.29 n.d. 0.3 0.16 n.d.
1.90 0.07 2.26 3.38 n.d. 0.23 0.44 n.d.
Fig. 2. Expression of Arabidopsis CYP85A2 and rice CYP85A1 in yeast WAT11 cells. The coding sequences of the 2 cDNA were translationally fused with the V5 anti gene in the yeast expression vector pYES_DEST52. The cDNA corresponding to the transgenes were obtained as RT-PCR products, whereas the cDNA was not obtained from the vector control. Similarly, the proteins were successfully expressed and detected when the protein blot was hybridized with anti-V5 antibody.
Unit, ng/g fresh weight. n.d., not detected.
The retention times of the authentic compounds, namely, 6-deo xoCS, CS, and BL, as detected by gas chromatography–mass spec trometry (GC–MS) were 10.20, 11.12, and 11.85 min, respectively. When the control strain harboring an empty vector was fed with 6-deoxoCS, it produced peaks corresponding to the substrate without exhibiting any other peaks, possibly because of the back ground activity of the yeast strain. These results confirmed that yeasts are reliable for use as heterologous expression systems (Fig. 3A, row “vector”). In contrast, the AtCYP85A2-harboring strain yielded 3 distinguishable peaks corresponding to the substrate, CS, and BL (Fig. 3A, row “AtCYP85A2”). This peak pattern confirms that AtCYP85A2 acts as a BL synthase by converting 6-deoxoCS to BL via CS. Finally, rice OsCYP85A1-harboring strains were treated with 6deoxoCS; however, no peak corresponding to BL was discernable (Fig. 3A, row “OsCYP85A1”). Fig. 3B shows the results of the CS conversion tests. When the tests were performed on authentic CS and BL compounds, they were eluted in 11.13 and 11.85 min, respectively. Once again, the vector control failed to exhibit metabolism of the added CS even to a small extent; however, it was apparent that Arabidopsis CYP85A2 converted CS to BL. In contrast, OsCYP85A1 did not yield any peak corresponding to BL (Fig. 3B, row “OsCYP85A1”). These results suggest that neither 6-deoxoCS nor CS was converted to BL in the OsCYP85A1-harboring yeast cells. It is therefore likely that OsCYP85A1 does not possess BL synthase activity per se. ch ro m a to g ra p hy- m a s s
able level in rice. It is generally accepted that CT is specifically found in Catharanthus roseus [8]. Thus, the failure to detect CT in rice is not surprising. More importantly, BL was not produced at a detectable level in rice, possibly due to its extremely low endoge nous levels or due to the absence of BL synthase activity in rice. This failure to detect BL in rice may be attributable to a simple technical limitation. In general, BL occurs in extremely low concen trations in plants; consequently, it is not readily detectable even with advanced analytical methods [15]. The level of BL in Arabid opsis, tomato, and rice plants varied depending on the tissues examined [12,15,16]. Therefore, a more thorough analysis of the BR levels in specific tissues of rice may result in the detection of BL. However, BL was detected neither in the wild-type rice plants (Table 1) nor in the d61 (Osbri1) mutants, which are defective in the rice homolog of the BR receptor [11,12]. Previously, we have demonstrated that Arabidopsis mutants that are defective in BR signaling, such as bri1-5 and bin2/dwf121D, accumulate significant amounts of BL due to the uncoupling of feedback downregulation from the BR signal transduction path way [15,17]. Thus, the failure to detect endogenous BL in rice d61 mutants strongly suggests the absence of BL in rice rather than a simple technical limitation in detection. This hypothesis is sup ported by the observation that none of the 8 species belonging to the Gramineae family was found to possess BL, whereas all these eight species endogenously express CS, which is the penultimate compound in BR biosynthesis [1].
(GC-MS)
8
Cloning and heterologous expression of Arabidopsis CYP85A2 and rice CYP85A1 genes in yeast It has been reported that 6-deoxoCS is converted to BL via CS (Fig. 1) [9,18,19]. Since both Arabidopsis CYP85A2 and tomato CYP85A3 have both been reported to be BL synthases, we analyzed the rice homolog of CYP85A to determine if this also possessed BL synthase activity. Fig. 2A shows that the transgenes were successfully expressed such that the mRNAs were clearly detectable by RT-PCR analysis. Furthermore, the proteins translated from the mRNA were all detectable by protein gel-blot analysis. Metabolic conversion of 6-deoxoCS and CS by CYP85 enzymes An analysis of metabolic conversion was performed using yeast strains harboring Arabidopsis CYP85A2 or rice CYP85A1 after feeding the strains with 6-deoxoCS, which is a known substrate for these CYP85 enzymes. Fig. 3A summarizes the results of this analysis.
Repeated analyses yield the same results In order to increase the accuracy of the results obtained, we replicated this experiment. Table 2 summarizes the results of this replicate. When 6-deoxoCS was administered to the AtCYP85A2 control cells, the majority of the substrate was converted to CS or BL; the exogenously supplied substrate, 6-deoxoCS, represented only 2% on average of the total recovered BRs, whereas the CS and BL levels represented 94% and 5%, respectively. A 94% CS fraction confirmed that AtCYP85A2 functions mainly as a CS synthase, and that it also yields BL; however, the conversion rate of CS to BL was substantially lower (5%). On the other hand, when CS was supplied as a substrate to AtCYP85A2, the majority of this substrate was not converted into BL; approximately 3% of the recovered BRs were identi fied as BL. Although AtCYP85A2 had weak enzymatic activity, we clearly demonstrated that it functions as a BL synthase and that the biochemical system we employed was sufficiently sen sitive to detect the biochemical function of AtCYP85A2 as a BL synthase. When 6-deoxoCS was administered to OsCYP85A1-harboring yeast cells, it was converted to CS; the average conversion rate
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
617
Fig. 3. Gas chromatography–mass spectrometry (GC–MS) results of the metabolic conversion analysis. Yeast strains harboring an empty vector, AtCYP85A2, and OsCYP85A1 were incubated with 6-deoxoCS (A) or CS (B); subsequently, the BRs were prepared and analyzed. The rows labeled “Authentic” indicate the peaks obtained when the authen tic compounds 6-deoxoCS, CS, or BL were analyzed by GC–MS. In a vector control, no obvious peaks except those corresponding to the supplied precursors are observed. AtCYP85A2-containing yeast cells successfully converted 6-deoxoCS to CS and BL, whereas OsCYP85A1 only metabolized 6-deoxoCS to CS. Similarly, CS was transformed into BL by AtCYP85A2; however, OsCYP85A1 failed to generate BL, suggesting that OsCTP85A1 lacks a functional BL synthase.
Table 2 Metabolic conversion of 6-deoxoCS or CS by OsCYP85A1 and AtCYP85A2 Feeding
6-DeoxoCS (5000 ng)
Recovery
6-DeoxoCS
CS (5000 ng) CS
BL
CS
BL
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
Empty vector
2100
100
n.d.
0
n.d.
0
2000
100
n.d.
0
AtCYP85A2
4 1 1 2
900 2400 3300 2200
90 96 95 94
60 70 170 100
6 3 5 5
1400 2800 1400 1867
95 98 97 97
70 60 50 60
5 2 3 3
OsCYP85A1
200 2600 1900 1567
18 65 63 58
900 1400 1100 1133
82 35 37 42
n.d. n.d. n.d. n.d.
0 0 0 0
2100 1400 1800 1767
100 100 100 100
n.d. n.d. n.d. n.d.
0 0 0 0
OsCYP85D3Ser
1400
54
1200
46
n.d.
0
2200
100
n.d.
0
40 30 20 30
Yeast strains harboring an empty vector, AtCYP85A2, OsCYP85A1, or OsCYP85A1-D3Ser constructs were supplied with substrates such as 6-deoxoCS and CS. After incubation with the substrates, BRs were recovered, and the level of each BR was determined using gas chromatography–mass spectrometry (GC–MS). For each recovered BR, the col umns labeled with “ng” represent the amount of compound recovered in nanograms, and “Fraction” indicates the fraction of the metabolites over the total amount of com pounds recovered. The average values obtained from 3 repeat experiments are shown in bold face.
was 42% (Table 2). BL, however, was not detected in any trials. The relatively lower conversion rate of 6-deoxoCS to CS obtained with OsCYP85A1 than that of Arabidopsis CYP85A2 suggests that the rice enzyme is less efficient in the enzymatic conversion reac tion. When we reexamined this enzymatic efficiency of CYP85A2
by administering an excess amount of CS, we could recover only the administered substrate; no trace amounts of BL were detected. A failure to detect any BL from the OsCYP85A1-trans formed cells confirms that OsCYP85A1 is devoid of BL synthase activity.
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Fig. 4. Multiple sequence alignment and phylogenetic relationships among the CYP85 proteins. The OsCYP85A1 sequence is distinguishable by the presence of 3 Ser residues at 162–164, and Pro at position 407 instead of Ser found in the known BL synthases: Arabidopsis-CYP85A2 and tomato-CYP85A3. The CYP85 proteins are primarily divided into 2 clades comprising the species of monocotyledonous and dicotyledonous plants (B). Monocotyledonous plant species possess a single copy of the CYP85 gene, whereas the dicotyledonous plants possess at least 2 copies. The length of the horizontal lines indicates the degree of differences in the amino acid sequences, and the numbers at the branching points represent bootstrap values obtained from 1000 replicate trees.
Independent evolution of the BL synthase genes in many plant species The overall amino acid sequence identity between OsCYP85A1 and the known BL synthase AtCYP85A2 is as high as 59%. How ever, OsCYP85A1 does not function as a BL synthase. There fore, we sought to determine how the OsCYP85A1 sequence differs from the primary structure of other BL-synthetic CYP85 proteins. Fig. 4A displays part of a multiple sequence alignment. We searched for amino acid residues that are conserved in BL synthases (AtCYP85A2 and LeCYP85A3) but differed in CS syn thases (OsCYP85A1, AtCYP85A1, and LeCYP85A1). The Pro resi due at position 407 of OsCYP85A1 satisfied this requirement; at this position, all the established BL synthases possessed Ser instead of the Pro found in rice and barley, and the Asn found in LeCYP85A1, PsCYP85A1, and AtCYP85A1. In addition to the Ser407, the rice amino acid sequence contains 3 additional Ser residues (Fig. 4A). In order to determine if the unusual insertion of the 3 Ser residues in the OsCYP85A1 sequence was respon sible for the elimination of its BL synthase function as well as the reduced CS synthase activity relative to AtCYP85A2, we per formed site-directed mutagenesis to delete these 3 additional amino acid residues; subsequently, we performed an analysis of metabolic conversion. As indicated in Table 2, the deleted detection of the construct OsCYP85A1-D3Ser resulted in a slight increase in CS synthase activity; however, it still failed to generate BL. The phylogenetic relationship among the CYP85 proteins was examined by constructing a phylogenetic tree (Fig. 4B). Many of the plant species were observed to possess more than 2 copies of the CYP85 gene: Pisum sativum (pea), Vitis vinifera (grape), Populus
trichocarpa (Populus), L. esculentum (tomato), and Arabidopsis. However, monocotyledonous plants, including barley, maize, Brac hypodium, and rice were observed to possess just a single copy of the CYP85 gene. When 2 copies of CYP85 were present in a species, they were clustered together in the tree. This implies that these genes multiplied independently in each species after speciation and did not descend from a duplicated ancestor. However, interest ingly, the 2 copies of CYP85 in Populus and grape are clustered inter specifically; this implies that these genes were duplicated before speciation. In conclusion, a point should be made that CS is as active enough to trigger the BR signal transduction pathways as BL. A rice lamina inclination bioassay revealed that CS successfully induces lamina bending of rice, although its effectiveness is slightly lesser than BL [2,4]. In addition, we have shown that an Arabidopsis mutant defective for the two CYP85s is rescued to wild-type phenotype by addition of CS [20]. These results sug gest that CS is indeed an active BR per se. Furthermore, it has been reported that Arabidopsis BR receptor BRI1 successfully binds to both CS and BL [21,22]. Therefore, it could be presumed that OsBRI1 also retains binding affinity to both of the two BRs based on its degree of sequence similarity with an Arabidopsis homolog. Absence of endogenous BL but presence of a capability to respond to BL better than CS in rice can be interpreted that BR biosynthetic and perception pathways may have evolved inde pendently in rice plants. Lyc o p e r s i c o n
Acknowledgments We thank Dr. Pompon for his gift of Yeast WAT11 and WAT21 cells. This research was supported, in part, by grants from the Plant Diversity Research Center of the 21st Century Frontier Research 21 st
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
Program funded by the Ministry of Education, Science and Technology of the Korean Government (PF06304-01), the Plant Metabolism Research Center at Kyung Hee University, Science Research Center Program from the Korea Science and Engineer ing Foundation, a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, a Basic Research Promotion Fund, KRF-2005-070-C00129) to S.C., and a Grant-in-Aid for Scien tific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.F. (Grant No. 19380069). B.K. was supported by a BK21 Research Fellowship from the Ministry of Education, Science and Technology. ( P F 0 6 3 0 4 - 01 ) ,
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[10] T. Nomura, T. Kushiro, T. Yokota, Y. Kamiya, G.J. Bishop, S. Yamaguchi, The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis, J. Biol. Chem. 280 (2005) 17873– 17879. [11] C. Yamamuro, Y. Ihara, X. Wu, T. Noguchi, S. Fujioka, S. Takatsuto, M. Ashikari, H. Kitano, M. Matsuoka, Loss of function of a rice brassinosteroid insensitive 1 homolog prevents internode elongation and bending of the lamina joint, Plant Cell 12 (2000) 1591–1606. [12] A. Nakamura, S. Fujioka, H. Sunohara, N. Kamiya, Z. Hong, Y. Inukai, K. Miura, S. Takatsuto, S. Yoshida, M. Ueguchi-Tanaka, Y. Hasegawa, H. Kitano, M. Mats uoka, The role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice, Plant Physiol. 140 (2006) 580–590. [13] D. Pompon, B. Louerat, A. Bronine, P. Urban, Yeast expression of animal and plant P450s in optimized redox environments, Methods Enzymol. 272 (1996) 51–64. [14] S. Fujioka, S. Takatsuto, S. Yoshida, An early C-22 oxidation branch in the bras sinosteroid biosynthetic pathway, Plant Physiol. 130 (2002) 930–939. [15] H.B. Kim, M. Kwon, H. Ryu, S. Fujioka, S. Takatsuto, S. Yoshida, C.S. An, I. Lee, I. Hwang, S. Choe, The regulation of DWARF4 expression is likely a critical mech anism in maintaining the homeostasis of bioactive brassinosteroids in Arabid opsis, Plant Physiol. 140 (2006) 548–557. [16] T. Montoya, T. Nomura, T. Yokota, K. Farrar, K. Harrison, J.D. Jones, T. Kaneta, Y. Kamiya, M. Szekeres, G.J. Bishop, Patterns of Dwarf expression and brassi nosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development, Plant J. 42 (2005) 262–269. [17] S. Choe, R.J. Schmitz, S. Fujioka, S. Takatsuto, M.O. Lee, S. Yoshida, K.A. Feld mann, F.E. Tax, Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3b-like kinase, Plant Physiol. 130 (2002) 1506–1515. [18] G. Bishop, T. Nomura, T. Yokota, T. Montoya, J. Castle, K. Harrison, T. Kushiro, Y. Kamiya, S. Yamaguchi, S. Bancos, A.M. Szatmari, M. Szekeres, Dwarfism and cytochrome P450-mediated C-6 oxidation of plant steroid hormones, Bio chem. Soc. Trans. 34 (2006) 1199–1201. [19] C.E. Jager, G.M. Symons, T. Nomura, Y. Yamada, J.J. Smith, S. Yamaguchi, Y. Kamiya, J.L. Weller, T. Yokota, J.B. Reid, Characterization of two brassinosteroid C-6 oxidase genes in pea, Plant Physiol. 143 (2007) 1894–1904. [20] M. Kwon, S. Fujioka, J.H. Jeon, H.B. Kim, S. Takatsuto, S. Yoshida, C.S. An, S. Choe, A double mutant for the CYP85A1 and CYP85A2 genes of Arabidopsis exhibits a brassinosteroid dwarf phenotype, J. Plant Biol. 48 (2005) 237–244. [21] T. Kinoshita, A. Cano-Delgado, H. Seto, S. Hiranuma, S. Fujioka, S. Yoshida, J. Chory, Binding of brassinosteroids to the extracellular domain of plant recep tor kinase BRI1, Nature 433 (2005) 167–171. [22] Z.Y. Wang, H. Seto, S. Fujioka, S. Yoshida, J. Chory, BRI1 is a critical component of a plasma-membrane receptor for plant steroids, Nature 410 (2001) 380–383.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c
Castasterone is a likely end product of brassinosteroid biosynthetic pathway in rice Bo Kyung Kim a, Shozo Fujioka b, Suguru Takatsuto c, Masafumi Tsujimoto b, Sunghwa Choe a,* a
Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea Laboratory of Cellular Biochemistry, RIKEN, Wako-shi, Saitama 351-0198, Japan c Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan b
a r t i c l e
i n f o
Article history: Received 9 July 2008 Available online 24 July 2008 Keywords: Brassinolide synthase Brassinosteroids CYP85 Cytochrome P450 Phytosteroids Steroid hormones Arabidopsis Rice
a b s t r a c t Brassinolide is known to be the most biologically active compound among more than 50 brassinosteroids identified to date. However, brassinolide has not been detected in rice. To determine if this is due to the lack of the brassinolide synthase function in the rice CYP85A enzyme, we performed analyses to study metabolic conversion using a yeast strain harboring the rice CYP85A1 gene. In repeated feeding tests where the substrates were used, the biosynthetic pathway progressed only up to the synthesis of castas terone, not of brassinolide. Phylogenetic analysis of the CYP85 amino acid sequences revealed that dupli cation of the CYP85 gene has occurred in most dicotyledonous plant genomes; further, 1 of the 2 copies of CYP85 is evolving to develop a brassinolide synthase function. However, only a single copy of this gene is found in the currently available genome sequences of graminaceous plants; this is a likely explanation for the absence of an endogenous pool of brassinolide in rice plants. © 2008 Elsevier Inc. All rights reserved.
Brassinosteroids (BRs) are a group of natural compounds that possess a steroid backbone with oxidized carbon atoms at multiple sites in the core ring and the side chain (Fig. 1). To date, more than 50 BR compounds have been identified from 61 plants across the plant kingdom [1]. Bioassays such as rice lamina inclination assay [2–4] proved brassinolide (BL), the most highly oxidized form among BRs, the most biologically active. Campesterol (CR) is the precursor of the BL biosynthetic pathway; this pathway is chan neled through 2 alternative routes depending on the order of 2 reactions, namely, C-22 hydroxylation and C-5 reduction. The C-22 hydroxylation reactions are mediated by the Arabidopsis thaliana DWARF4 protein, which belongs to the cytochrome P450 monooxy genase family CYP90B1 [5,6]. The resulting C-22 hydroxylated com pounds are then hydroxylated at the C-23 position by CYP90C1 and CYP90D1 [7] before being subjected to reduction at position C-5. Thereafter, these compounds undergo C-3 epimerization and C-2 hydroxylation reactions, and finally 3 consecutive C-6 oxidation reactions occur, leading to the generation of castasterone (CS) and BL [8]. BL is mainly derived from 6-deoxocastasterone (6-deoxoCS) by the catalytic actions of a group of cytochrome P450 enzymes belonging to the CYP85 family; these enzymes include Arabidopsis CYP85A2 (AtCYP85A2) and Lycopersicon esculentum CYP85A3
* Corresponding author. Fax: +82 2 872 1993. E-mail address: shc[email protected] (S. Choe). 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.07.073
(LeCYP85A3). These enzymes mediate 3 consecutive steps in the BL biosynthetic pathway: C-6 hydroxylation, C-6 dehydrogenation, and a Baeyer–Villiger-type oxidation to create a lactone at ring B of the steroid backbone (Fig. 1) [9,10]. The Arabidopsis genome possesses 2 copies of the CYP85 gene; due to the functional redun dancy between these 2 genes, plants exhibit conventional BR dwarf phenotype only when the functions of both the genes are simulta neously disrupted [9,10]. In contrast to the Arabidopsis and tomato genomes, the com pleted sequence of the rice genome revealed only 1 copy of the CYP85 gene. In addition, BL is not detectable even in Osbri1 mutant plants, which are assumed to accumulate large amounts of BRs in vivo [11,12]. To examine if the rice CYP85A1 (OsCYP85A1) enzyme functions as a CS synthase rather than both CS synthase and BL syn thase, we expressed the OsCYP85A1 gene in yeast and performed analyses to study metabolic conversion. B a eye r-V i l l i ge r- t y p e
Materials and methods Cloning and heterologous expression of AtCYP85A2 and OsCYP85A1. The cDNAs encoding AtCYP85A2 and OsCYP85A1 enzymes were cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) performed on the mRNA isolated from Arabidopsis and rice seedlings. The oligonucleotide sequences are as follows: OsCYP85-F, 59-CACCATGGTGTTGGTGGC-39; OsCYP85R, 59-GCAGTAATCTTGAACGCGGATAT-39; AtCYP85A2-F, 59-CACC ATGATGATGATTTTGGGT-39; and AtCYP85A2-R, 59-GCAGTAAG t ra n s c r i pta s e – p o ly m e ra s e
5 9 - CAC CATGATGATGAT T T TG G GT- 3 9 ;
5 9 - G CAGTA AG GTGA ACAC T TA AG - 3 9 .
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
615
OH OH OH OH
HO
1 AtCYP85A1 AtCYP85A2 LeCYP85A1 LeCYP85A3 PsCYP85A1 PsCYP85A6 OsCYP85A1
H
6-Deoxotyphasterol (6-DeoxoTY) PsCYP92A6
HO
H
O
Typhasterol (TY) OH
OH OH OH
HO
1
HO
HO HO
H
H
6-Deoxocastasterone (6-DeoxoCS)
Castasterone (CS)
O
AtCYP85A2 LeCYP85A3
OH
2
OH
HO
HO H
O
Brassinolide (BL)
O
Fig. 1. Part of the brassinolide biosynthetic pathway. The pathway precursor, campesterol, is converted to 6-deoxoTY via a series of reactions mediated by CYP90 enzymes and steroid 3-dehydrogenases. The carbon at the C-6 position of 6-deoxoTY and 6-deoxoCS is oxidized by CYP85 proteins to yield TY and CS, respectively (Reaction 1). Finally, CS is further oxidized to BL by the BL synthase action of CYP85 enzymes such as Arabidopsis CYP85A2 and tomato CYP85A3 (Reaction 2).
GTGAACACTTAAG-39. The cDNA fragments were subcloned into a pYES_DEST52 yeast expression vector (Invitrogen, USA) containing a uracil (URA) selection marker according to the protocol provided by the manufacturer. The cloned vector was transformed into the WAT11 strain for optimized expression of plant P450 proteins [13]. The selected transformants were subcultured and their respiratory capability was tested by observing their growth on glycerol-con taining N3 medium according to the previously described proto cols [14]. Site-specific mutagenesis was performed by PCR using overlap ping nucleotides containing the designated mutations: FOR, 59-tc gccgcctggtcctcctccgccgtcgtcgacatccaggc-39; and REV, 59-cctggatgt cgacgacggcggaggaggaccaggcggcga-39. Deletion of the 3 target Ser residues was confirmed by DNA sequencing using the M13 primer located in the expression vector. Quantitative analysis of sterols and BRs using gas chromatogra phy-mass spectrometry. The endogenous levels of both sterols and BRs were determined using 4-month-old rice plants according to the methods described previously [14]. In case of Arabidopsis, the levels in ‘‘Wassilewskija-2 (Ws-2)” ecotype was similarly deter mined using the aerial parts of 5-week-old adult plants grown in soil under regular long day conditions (16:8 h, light:dark). Protein gel-blot analysis. To check whether the transgene was correctly expressed, the total proteins were isolated from the yeast cells, and 20 lg of each protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Pro teins resolved by SDS–PAGE were transferred to nitrocellulose membranes (Amersham Bioscience, USA), and the protein bands were visualized by immunoblotting followed by incubation with anti-V5 antibodies (Invitrogen, USA) conjugated to horseradish per oxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnol ogy, USA). i n ‘ ‘ Wa s s i l ews k ij a - 2 ( W s - 2 ) ’ ’
( 16 h : 8
l i g h t: d a rk ) .
s u l fa te - p o lya c r yl a m i d e
Multiple sequence alignment and phylogenetic analysis. To study the distribution of CYP85 genes in plants, the GenBank database was searched using the rice CYP85A1 amino acid sequence as a probe. Sequences sharing greater than 55% identity were selected for fur ther comparison. Since full-length cDNA sequences for maize and Brachypodium were not available, virtual cDNA sequences were constructed using the Brachypodium shotgun sequences released by the Joint Genome Institute (JGI) covering the Brachypodium genome to a depth of 4£ and expressed sequence tags (ESTs) before obtaining the deduced amino acid sequences. The data base accession numbers for the CYP85 protein sequences are as follows: NP001050623 (rice), ABH01181 (barley), BAF56235 (pea; CYP85A1), BAF56236 (pea; CYP85A6), ABB60086 (grape; CYP85A1), CAO41843 (grape; CYP85A6), Q43147 (tomato; CYP85A1), Q50LE0 (tomato; CYP85A3), Q940V4 (Arabidopsis; CYP85A2), and Q9FMA5 (Arabidopsis; CYP85A1). Sequences for Populus-CYP85A1 and Popu lus-CYP85A4 were obtained from the cytochrome P450 database (http://drnelson.utmem.edu/CytochromeP450.html). A phylogenetic tree was constructed based on the full-length deduced amino acid sequences by using the neighbor-joining method of the CLUSTALW program (Version 1.83). Bootstrap mode (1000 replications) was used to estimate the confidence level that can be assigned to the branching nodes in the tree. full length
( 1, 0 0 0
Results and discussion
( S D S - PAG E )
S D S - PAG E
The endogenous level of BL is not detectable in rice Table 1 summarizes the endogenous BR levels in rice and Arabid opsis; the data presented in this table indicate that all the sterols that serve as precursors of BR biosynthesis were detected in the study. However, 2 BRs—CT and BL—were not produced at a detect
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Table 1 Endogenous levels of sterols and BRs in the rice cultivar “Dongjin” and Arabidopsis “Wassilewskija-2” wild-type Rice
Arabidopsis
Sterols 24-Methylenecholesterol (24-MC) Campesterol (CR) Campestanol (CN) 6-Oxocampestanol (6-OxoCN)
2490 35,100 823 10.2
5340 35,300 978 31.6
BRs 6-Deoxocathasterone (6-DeoxoCT) 6-Deoxoteasterone (6-DeoxoTE) 6-Deoxotyphasterol (6-DeoxoTY) 6-Deoxocastasterone (6-DeoxoCS) Cathasterone (CT) Typhasterol (TY) Castasterone (CS) Brassinolide (BL)
0.42 0.04 2.9 0.29 n.d. 0.3 0.16 n.d.
1.90 0.07 2.26 3.38 n.d. 0.23 0.44 n.d.
Fig. 2. Expression of Arabidopsis CYP85A2 and rice CYP85A1 in yeast WAT11 cells. The coding sequences of the 2 cDNA were translationally fused with the V5 anti gene in the yeast expression vector pYES_DEST52. The cDNA corresponding to the transgenes were obtained as RT-PCR products, whereas the cDNA was not obtained from the vector control. Similarly, the proteins were successfully expressed and detected when the protein blot was hybridized with anti-V5 antibody.
Unit, ng/g fresh weight. n.d., not detected.
The retention times of the authentic compounds, namely, 6-deo xoCS, CS, and BL, as detected by gas chromatography–mass spec trometry (GC–MS) were 10.20, 11.12, and 11.85 min, respectively. When the control strain harboring an empty vector was fed with 6-deoxoCS, it produced peaks corresponding to the substrate without exhibiting any other peaks, possibly because of the back ground activity of the yeast strain. These results confirmed that yeasts are reliable for use as heterologous expression systems (Fig. 3A, row “vector”). In contrast, the AtCYP85A2-harboring strain yielded 3 distinguishable peaks corresponding to the substrate, CS, and BL (Fig. 3A, row “AtCYP85A2”). This peak pattern confirms that AtCYP85A2 acts as a BL synthase by converting 6-deoxoCS to BL via CS. Finally, rice OsCYP85A1-harboring strains were treated with 6deoxoCS; however, no peak corresponding to BL was discernable (Fig. 3A, row “OsCYP85A1”). Fig. 3B shows the results of the CS conversion tests. When the tests were performed on authentic CS and BL compounds, they were eluted in 11.13 and 11.85 min, respectively. Once again, the vector control failed to exhibit metabolism of the added CS even to a small extent; however, it was apparent that Arabidopsis CYP85A2 converted CS to BL. In contrast, OsCYP85A1 did not yield any peak corresponding to BL (Fig. 3B, row “OsCYP85A1”). These results suggest that neither 6-deoxoCS nor CS was converted to BL in the OsCYP85A1-harboring yeast cells. It is therefore likely that OsCYP85A1 does not possess BL synthase activity per se. ch ro m a to g ra p hy- m a s s
able level in rice. It is generally accepted that CT is specifically found in Catharanthus roseus [8]. Thus, the failure to detect CT in rice is not surprising. More importantly, BL was not produced at a detectable level in rice, possibly due to its extremely low endoge nous levels or due to the absence of BL synthase activity in rice. This failure to detect BL in rice may be attributable to a simple technical limitation. In general, BL occurs in extremely low concen trations in plants; consequently, it is not readily detectable even with advanced analytical methods [15]. The level of BL in Arabid opsis, tomato, and rice plants varied depending on the tissues examined [12,15,16]. Therefore, a more thorough analysis of the BR levels in specific tissues of rice may result in the detection of BL. However, BL was detected neither in the wild-type rice plants (Table 1) nor in the d61 (Osbri1) mutants, which are defective in the rice homolog of the BR receptor [11,12]. Previously, we have demonstrated that Arabidopsis mutants that are defective in BR signaling, such as bri1-5 and bin2/dwf121D, accumulate significant amounts of BL due to the uncoupling of feedback downregulation from the BR signal transduction path way [15,17]. Thus, the failure to detect endogenous BL in rice d61 mutants strongly suggests the absence of BL in rice rather than a simple technical limitation in detection. This hypothesis is sup ported by the observation that none of the 8 species belonging to the Gramineae family was found to possess BL, whereas all these eight species endogenously express CS, which is the penultimate compound in BR biosynthesis [1].
(GC-MS)
8
Cloning and heterologous expression of Arabidopsis CYP85A2 and rice CYP85A1 genes in yeast It has been reported that 6-deoxoCS is converted to BL via CS (Fig. 1) [9,18,19]. Since both Arabidopsis CYP85A2 and tomato CYP85A3 have both been reported to be BL synthases, we analyzed the rice homolog of CYP85A to determine if this also possessed BL synthase activity. Fig. 2A shows that the transgenes were successfully expressed such that the mRNAs were clearly detectable by RT-PCR analysis. Furthermore, the proteins translated from the mRNA were all detectable by protein gel-blot analysis. Metabolic conversion of 6-deoxoCS and CS by CYP85 enzymes An analysis of metabolic conversion was performed using yeast strains harboring Arabidopsis CYP85A2 or rice CYP85A1 after feeding the strains with 6-deoxoCS, which is a known substrate for these CYP85 enzymes. Fig. 3A summarizes the results of this analysis.
Repeated analyses yield the same results In order to increase the accuracy of the results obtained, we replicated this experiment. Table 2 summarizes the results of this replicate. When 6-deoxoCS was administered to the AtCYP85A2 control cells, the majority of the substrate was converted to CS or BL; the exogenously supplied substrate, 6-deoxoCS, represented only 2% on average of the total recovered BRs, whereas the CS and BL levels represented 94% and 5%, respectively. A 94% CS fraction confirmed that AtCYP85A2 functions mainly as a CS synthase, and that it also yields BL; however, the conversion rate of CS to BL was substantially lower (5%). On the other hand, when CS was supplied as a substrate to AtCYP85A2, the majority of this substrate was not converted into BL; approximately 3% of the recovered BRs were identi fied as BL. Although AtCYP85A2 had weak enzymatic activity, we clearly demonstrated that it functions as a BL synthase and that the biochemical system we employed was sufficiently sen sitive to detect the biochemical function of AtCYP85A2 as a BL synthase. When 6-deoxoCS was administered to OsCYP85A1-harboring yeast cells, it was converted to CS; the average conversion rate
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
617
Fig. 3. Gas chromatography–mass spectrometry (GC–MS) results of the metabolic conversion analysis. Yeast strains harboring an empty vector, AtCYP85A2, and OsCYP85A1 were incubated with 6-deoxoCS (A) or CS (B); subsequently, the BRs were prepared and analyzed. The rows labeled “Authentic” indicate the peaks obtained when the authen tic compounds 6-deoxoCS, CS, or BL were analyzed by GC–MS. In a vector control, no obvious peaks except those corresponding to the supplied precursors are observed. AtCYP85A2-containing yeast cells successfully converted 6-deoxoCS to CS and BL, whereas OsCYP85A1 only metabolized 6-deoxoCS to CS. Similarly, CS was transformed into BL by AtCYP85A2; however, OsCYP85A1 failed to generate BL, suggesting that OsCTP85A1 lacks a functional BL synthase.
Table 2 Metabolic conversion of 6-deoxoCS or CS by OsCYP85A1 and AtCYP85A2 Feeding
6-DeoxoCS (5000 ng)
Recovery
6-DeoxoCS
CS (5000 ng) CS
BL
CS
BL
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
ng
Fraction (%)
Empty vector
2100
100
n.d.
0
n.d.
0
2000
100
n.d.
0
AtCYP85A2
4 1 1 2
900 2400 3300 2200
90 96 95 94
60 70 170 100
6 3 5 5
1400 2800 1400 1867
95 98 97 97
70 60 50 60
5 2 3 3
OsCYP85A1
200 2600 1900 1567
18 65 63 58
900 1400 1100 1133
82 35 37 42
n.d. n.d. n.d. n.d.
0 0 0 0
2100 1400 1800 1767
100 100 100 100
n.d. n.d. n.d. n.d.
0 0 0 0
OsCYP85D3Ser
1400
54
1200
46
n.d.
0
2200
100
n.d.
0
40 30 20 30
Yeast strains harboring an empty vector, AtCYP85A2, OsCYP85A1, or OsCYP85A1-D3Ser constructs were supplied with substrates such as 6-deoxoCS and CS. After incubation with the substrates, BRs were recovered, and the level of each BR was determined using gas chromatography–mass spectrometry (GC–MS). For each recovered BR, the col umns labeled with “ng” represent the amount of compound recovered in nanograms, and “Fraction” indicates the fraction of the metabolites over the total amount of com pounds recovered. The average values obtained from 3 repeat experiments are shown in bold face.
was 42% (Table 2). BL, however, was not detected in any trials. The relatively lower conversion rate of 6-deoxoCS to CS obtained with OsCYP85A1 than that of Arabidopsis CYP85A2 suggests that the rice enzyme is less efficient in the enzymatic conversion reac tion. When we reexamined this enzymatic efficiency of CYP85A2
by administering an excess amount of CS, we could recover only the administered substrate; no trace amounts of BL were detected. A failure to detect any BL from the OsCYP85A1-trans formed cells confirms that OsCYP85A1 is devoid of BL synthase activity.
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Fig. 4. Multiple sequence alignment and phylogenetic relationships among the CYP85 proteins. The OsCYP85A1 sequence is distinguishable by the presence of 3 Ser residues at 162–164, and Pro at position 407 instead of Ser found in the known BL synthases: Arabidopsis-CYP85A2 and tomato-CYP85A3. The CYP85 proteins are primarily divided into 2 clades comprising the species of monocotyledonous and dicotyledonous plants (B). Monocotyledonous plant species possess a single copy of the CYP85 gene, whereas the dicotyledonous plants possess at least 2 copies. The length of the horizontal lines indicates the degree of differences in the amino acid sequences, and the numbers at the branching points represent bootstrap values obtained from 1000 replicate trees.
Independent evolution of the BL synthase genes in many plant species The overall amino acid sequence identity between OsCYP85A1 and the known BL synthase AtCYP85A2 is as high as 59%. How ever, OsCYP85A1 does not function as a BL synthase. There fore, we sought to determine how the OsCYP85A1 sequence differs from the primary structure of other BL-synthetic CYP85 proteins. Fig. 4A displays part of a multiple sequence alignment. We searched for amino acid residues that are conserved in BL synthases (AtCYP85A2 and LeCYP85A3) but differed in CS syn thases (OsCYP85A1, AtCYP85A1, and LeCYP85A1). The Pro resi due at position 407 of OsCYP85A1 satisfied this requirement; at this position, all the established BL synthases possessed Ser instead of the Pro found in rice and barley, and the Asn found in LeCYP85A1, PsCYP85A1, and AtCYP85A1. In addition to the Ser407, the rice amino acid sequence contains 3 additional Ser residues (Fig. 4A). In order to determine if the unusual insertion of the 3 Ser residues in the OsCYP85A1 sequence was respon sible for the elimination of its BL synthase function as well as the reduced CS synthase activity relative to AtCYP85A2, we per formed site-directed mutagenesis to delete these 3 additional amino acid residues; subsequently, we performed an analysis of metabolic conversion. As indicated in Table 2, the deleted detection of the construct OsCYP85A1-D3Ser resulted in a slight increase in CS synthase activity; however, it still failed to generate BL. The phylogenetic relationship among the CYP85 proteins was examined by constructing a phylogenetic tree (Fig. 4B). Many of the plant species were observed to possess more than 2 copies of the CYP85 gene: Pisum sativum (pea), Vitis vinifera (grape), Populus
trichocarpa (Populus), L. esculentum (tomato), and Arabidopsis. However, monocotyledonous plants, including barley, maize, Brac hypodium, and rice were observed to possess just a single copy of the CYP85 gene. When 2 copies of CYP85 were present in a species, they were clustered together in the tree. This implies that these genes multiplied independently in each species after speciation and did not descend from a duplicated ancestor. However, interest ingly, the 2 copies of CYP85 in Populus and grape are clustered inter specifically; this implies that these genes were duplicated before speciation. In conclusion, a point should be made that CS is as active enough to trigger the BR signal transduction pathways as BL. A rice lamina inclination bioassay revealed that CS successfully induces lamina bending of rice, although its effectiveness is slightly lesser than BL [2,4]. In addition, we have shown that an Arabidopsis mutant defective for the two CYP85s is rescued to wild-type phenotype by addition of CS [20]. These results sug gest that CS is indeed an active BR per se. Furthermore, it has been reported that Arabidopsis BR receptor BRI1 successfully binds to both CS and BL [21,22]. Therefore, it could be presumed that OsBRI1 also retains binding affinity to both of the two BRs based on its degree of sequence similarity with an Arabidopsis homolog. Absence of endogenous BL but presence of a capability to respond to BL better than CS in rice can be interpreted that BR biosynthetic and perception pathways may have evolved inde pendently in rice plants. Lyc o p e r s i c o n
Acknowledgments We thank Dr. Pompon for his gift of Yeast WAT11 and WAT21 cells. This research was supported, in part, by grants from the Plant Diversity Research Center of the 21st Century Frontier Research 21 st
B.K. Kim et al. / Biochemical and Biophysical Research Communications 374 (2008) 614–619
Program funded by the Ministry of Education, Science and Technology of the Korean Government (PF06304-01), the Plant Metabolism Research Center at Kyung Hee University, Science Research Center Program from the Korea Science and Engineer ing Foundation, a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, a Basic Research Promotion Fund, KRF-2005-070-C00129) to S.C., and a Grant-in-Aid for Scien tific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.F. (Grant No. 19380069). B.K. was supported by a BK21 Research Fellowship from the Ministry of Education, Science and Technology. ( P F 0 6 3 0 4 - 01 ) ,
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