Recent discoveries from brassinosteroid-deficient mutants led to the recognition that plants, like animals, use steroids to regulate their growth and development.
We describe the characterization of one member of a Brassica napus sulfotransferase gene family coding for an enzyme that catalyzes the O -sulfonation of brassinosteroids and of mammalian estrogenic steroids. The enzyme is specific for the hydroxyl group at position 22 of brassinosteroids with a preference for epicathasterone, an intermediate in the biosynthesis of epibrassinolide.
Enzymatic sulfonation of epibrassinolide abolishes its biological activity in the bean second internode bioassay. This mechanism of hormone inactivation by sulfonation is similar to the modulation of estrogen biological activity observed in mammals.
Furthermore, the expression of the B. This pattern of expression suggests that, in addition to an increased synthesis of proteins having antimicrobial properties, plants respond to pathogen infection by modulating steroid-dependent growth and developmental processes. Many developmental and physiological processes in organisms ranging from fungi to humans are regulated by a small number of steroid hormones.
However, until recently the plant kingdom was almost completely excluded from the field of steroid endocrinology. The recent demonstration that the Arabidopsis de-etiolated2 det2 and the constitutive photomorphogenesis and dwarfism cpd mutants are defective in the synthesis of brassinosteroids BRs 1 focused attention toward the physiological function of steroids in plants 1 , 2.
Since these initial discoveries, several other mutants impaired in BR synthesis or perception have been characterized at the molecular level BRs have been shown to elicit a broad spectrum of responses including the promotion of cell elongation and cell division, inhibition of de-etiolation in the dark, repression of light-regulated genes in the dark, and repression of stress-regulated genes 2 , 6 , 7.
Brassinolide was the first BR isolated and characterized from plants Fig. Since this initial discovery, the structure of over 40 analogues of brassinolide has been elucidated, and BRs are believed to be ubiquitous in the plant kingdom 9.
In view of their structural similarities with animal steroids, it has been proposed that BRs might interact with a soluble receptor in order to regulate the expression of specific genes Fig. This hypothesis has recently been challenged with the molecular characterization of the Brassinosteroid-Insensitive-1 BRI1 gene that encodes a putative BR receptor 4. The BRI1 protein belongs to a family of plant receptor-like transmembrane kinases. Despite this important discovery, direct or indirect binding of BRs to the BRI1 receptor has not been demonstrated, and their mode of action remains to be characterized.
In mammals, it is well recognized that sulfotransferases STs play an important role in the modulation of the biological activity of a number of compounds, such as steroid and thyroid hormones, and catecholamine neurotransmitters Sulfonate conjugation not only facilitates transport and excretion of hydrophobic molecules by increasing their water solubility, it abolishes the biological activity of hormones, such as estrogens.
Considering the important role of mammalian STs in the modulation of the biological activity of hormones, our research interest is to elucidate the function of plant STs in growth and development. In this paper, we describe the characterization of one member of a Brassica napus ST gene family coding for an enzyme that sulfonates BRs as well as mammalian estrogenic steroids. We also demonstrate that enzymatic sulfonation of epibrassinolide abolishes its biological activity.
In addition, our results indicate that the pattern of expression of the B. This represents the first example where the function of a PR protein can be linked to the modulation of the biological activity of a plant hormone by conjugation. Bacterial cells were collected by centrifugation, resuspended in 50 m m sodium phosphate buffer, pH 8. The recombinant BNST3 recovered in the soluble fraction was purified by affinity chromatography onto nickel-nitrilotriacetic acid-agarose matrix as recommended Qiagen.
Westar and Arabidopsis thaliana ecotype Columbia were grown in vermiculite under continuous white light. The roots of 7-day-old B. Northern blot analysis of total RNA was achieved under high stringency conditions according to standard procedures 16 using the 32 P-labeled coding region of BNST1 as a probe. The antibodies were found to cross-react with BNST2 and -3 data not shown. Analysis of substrate specificity was performed by testing enzymatic activity with three different concentrations of acceptor substrate as follows: Concentrations of epicathasterone were 2, 1, 0.
Stock solutions 1 m m of acceptor substrates were prepared in dimethyl sulfoxide or methanol. Commercial substrates were obtained from Sigma, and desulfoglucosinolates were kindly provided by D.
Incorporation of the 35 S-labeled sulfate was monitored as described Kinetic parameters were deduced from double-reciprocal Lineweaver-Burk plots.
The length of the second internode was measured 4 days following the application of test compounds. Brassinolide was purchased from Beak Technologies Inc.
The following BRs were synthesized from ergosterol using published procedures: A mixture of epicathasterone After cooling the mixture was diluted with HCl 10 ml, 1. Usual workup gave a residue, which was purified by flash chromatography on silica gel. The 1 H NMR data are in agreement with the given structure. A clone encoding a putative ST of unknown substrate specificity has recently been isolated from an A.
The BNST1, -2, and -3 genes belong to a multigene family comprising at least 12 members, as determined by Southern blot analysis and reverse transcriptase-polymerase chain reaction experiments data not shown. The variations among the three amino acid sequences are mostly observed in the domain that is involved in ST acceptor substrate recognition, suggesting that the three enzymes may have distinct substrate specificities Thus, it is likely that the B.
In contrast, the B. Comparison of the deduced amino acid sequences of B. Dots indicate gaps introduced for optimal alignment. Dashes indicate identical amino acids. Residues critical for catalysis and PAPS binding are indicated by arrows Domain II involved in the determination of substrate specificity is underlined However, watering the seedlings with 10 m m SA resulted in a rapid induction of their expression.
Due to their high level of nucleotide and amino acid sequence identity, analysis of the expression of BNSTs using the full-length BNST1 coding sequence, or polyclonal antibodies raised against the BNST1 protein as probes, should identify most, if not all, of the related expressed BNSTs. Therefore, the detection of several proteins in Western blots is consistent with BNST1, -2, and -3 being members of a multigene family.
In contrast, the detection of a single polypeptide with an apparent molecular mass of This is further supported by the pattern of expression of RaR in A. The blot was hybridized with the coding region of the BNST1 gene. B, same blot as shown in A hybridized with an actin probe. C, Western analysis of ST gene expression in B.
Total proteins were extracted from B. The membrane was incubated with anti-BNST1 antibodies. Size of molecular weight markers is indicated on the left. The choice of BNST3 was dictated by the fact that it is expressed in SA-treated seedlings as determined by reverse transcriptase-polymerase chain reaction and is the most similar to A. Although a variety of acceptor molecules of plant and mammalian origin e. It seems that a hydroxyl group attached to a rigid ring system, in estradiol, may limit its sulfonate accepting ability.
Substrate-dependent stereoselectivity has also been reported for the rat hydroxysteroid ST STa and phenol ST AST IV and was shown to be caused by the degree of steric bulk on the hydroxyl-containing chiral carbon 27 , Of those, BNST3 exhibits the highest affinity for epicathasterone followed by epiteasterone with apparent K m values of 1.
Furthermore, epicathasterone is the only substrate for which a significant enzymatic activity is measured at a concentration of 0. Although BNST3 also accepts epibrassinolide as a substrate, this activity is only observed at high substrate concentration Table I. The enzyme is also sensitive to the level of oxidation of ring B, as revealed by the low relative activity values obtained with 6-deoxoepicastasterone and 6-deoxoepicathasterone as substrates.
The most striking feature of BNST3 substrate specificity is its efficient use of the natural substrate epibrassinolide and the synthetic enantiomer 22 S ,23 S homobrassinolide, whereas it is inactive with the natural substrate brassinolide Table I. The fact that the enzyme accepts epicathasterone and epiteasterone but does not accept the synthetic substrate deoxyepiteasterone suggests strongly that sulfonation is taking place at position 22 of the steroid side chain Table I.
The specificity of the enzyme for position 22 explains why early BR intermediates and BR precursors lacking a hydroxyl group at this position are not accepted by the enzyme. It has previously been reported that hydroxylation of brassinosteroid intermediates at position 22 increases considerably their biological activity, suggesting that sulfonation at this position by BNST3 might lead to the loss of their function To test this hypothesis, we compared the ability of epibrassinolide and its sulfonated derivative to promote growth of the bean second internode, a well established biological test for the activity of BRs They belong to a gene family comprising at least 12 members having a high level of sequence identity with a previously characterized cDNA clone RaR from A.
Therefore, they represent the first characterized members of the new SULT4 family. When expressed in E. Whereas several studies 31 , 32 described the presence in plants of the mammalian steroids estradiol, estrone, and testosterone, as well as their physiological effects on growth, development, and flowering, the functional significance of estrogen sulfonation by a plant enzyme remains to be elucidated. Despite the lack of information on the accumulation of epimers of BRs in B.
These results call for a thorough investigation of BRs in B. BNST3 has the highest affinity for epicathasterone, of the substrates tested. The preference of BNST3 for an early precursor, as compared with the final product epibrassinolide, is quite surprising considering the fact that the latter is about fold more active However, cathasterone is considered to be the first BR precursor with significant biological activity, and it has been proposed that its synthesis is the rate-limiting step in the formation of the more potent BRs 5 , The substrate preference of BNST3 for epicathasterone may be an efficient mechanism to block its intrinsic activity and at the same time interfere with the biosynthesis of the more potent final product epibrassinolide.
The fact that the currently used techniques for the analysis of BRs are not suitable for the preservation of the labile sulfate moiety may explain the lack of reports on the characterization of sulfated BRs in plant extracts. However, it has been shown that rice seedlings administered labeled cathasterone produce non-glycosidic polar BR derivatives that may in fact be sulfate esters 34 , based on their chromatographic behavior and their susceptibility to solvolysis.
Our results also demonstrate that sulfonation of epibrassinolide abolishes its biological activity in the bean second internode bioassay suggesting that plants, like mammals, may modulate the biological activity of steroids by sulfonation. The recent finding that BRI1 codes for a potential transmembrane leucine-rich repeat-receptor kinase, which is required for BR perception, suggests that BRs mediate their biological activity on the external surface of the cell membrane 4.
Furthermore, the fact that the expression of the CPD gene is confined to specific cell types of leaves, other than those involved in elongation, suggests that BRs must be transported from source to target tissues Therefore, the sulfonation of BRs may interfere with their excretion, their transport, or their binding to the BRI1 receptor.
The results of several experiments suggest that BRs stimulate plant growth by increasing the plasticity of the cell wall In the context of a pathogen infection, this growth mechanism is undesirable since it reduces the protection offered by the cell wall.
The sulfonation of BRs by the SA- and pathogen-inducible steroid STs provides a mechanism through which BR-dependent cell wall hydrolysis might be stopped. Additional mechanisms are likely to parallel steroid ST activity to reduce the levels of biologically active BRs during pathogen infection. Furthermore, suppression of phytosterol biosynthesis following elicitor treatment has been reported in parsley, potato, tobacco, and Tabernaemontana divaricata