Luc Varin

Biochemical and Molecular Characterization of a Hydroxyjasmonate Sulfotransferase from Arabidopsis thaliana

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with K m values of 50 and 10 μm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring inA. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.

Sulphated flavonoids: an update

Abstract The increasing knowledge of various aspects of flavonoid sulphates calls for an update of previous reviews. This article describes the recent advances in their structural variation and distribution patterns in plants. The methods used in their photochemical analysis and structural determination are outlined. Furthermore, the recently developed chemical and enzymatic methods for the synthesis of specifically sulphated flavonoids are reviewed, and two hitherto unreported, position-specific flavonol sulphotransferases are described.

Inactivation of Brassinosteroid Biological Activity by a Salicylate-inducible Steroid Sulfotransferase from Brassica napus

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 napussulfotransferase gene family coding for an enzyme that catalyzes theO-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 24-epicathasterone, an intermediate in the biosynthesis of 24-epibrassinolide. Enzymatic sulfonation of 24-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. napussteroid sulfotransferase genes was found to be induced by salicylic acid, a signal molecule in the plant defense response. 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.

Sulfation and sulfotransferases 6: Biochemistry and molecular biology of plant sulfotransferases.

It is now well established that, in mammals, sulfate conjugation constitutes an important reaction in the transformation of xenobiotics and in the modulation of the biological activity of steroid hormones and neurotransmitter (1,2). The presence of a sulfate group on some molecules can also be a prerequisite for their biological function. For example, it is well known that the sulfate groups are directly involved in the molecular interaction between heparin and antithrombin III (3). In plants, sulfation also seems to play an important role in the intermolecular recognition and signaling processes, as indicated by the requirement of a sulfate moiety for the biological activity of gallic acid glucoside sulfate in the seismonastic and gravitropic movements of plants (4), and of Nod RM1 in the cortical cell division during early nodule initiation in Rhizobium meliloti‐alfalfainteraction (5). In addition, recent studies indicate that flavonoid conjugates, including the sulfate esters, may play a role in the regulation of plant growth by strongly binding the naphthylphthalamic acid receptor, thus blocking the quercetin‐stimulated accumulation of the auxin phytohormone (6). Although several sulfated metabolites are known to accumulate in a variety of plant species (7), the study of enzymes that catalyze the sulfation reaction in plants lagged considerably compared to those conducted with their mammalian homologs. This apparent lack of interest may have been because the function of plant‐sulfated metabolites is difficult to predict, since their accumulation is often restricted to a limited number of species. Despite this limitation, several plant sulfotransferases (STs) have been characterized at the biochemical level, and the cDNA clones encoding six plant STs have been isolated. Based on sequence homology, the plant ST coding sequences are grouped under the SULT3 family, also known as the flavonol ST family. This review summarizes our current knowledge of the plant STs and focuses on the functional significance of the sulfate conjugation in plant growth, development, and adaptation to stress.—Varin, L., Marsolais, F., Richard, M., Rouleau, M. Biochemistry and molecular biology of plant sulfotransferases. FASEB J. 11, 517–525 (1997)

Molecular and biochemical characterization of two brassinosteroid sulfotransferases from Arabidopsis, AtST4a (At2g14920) and AtST1 (At2g03760).

Mammalian sulfotransferases (EC 2.8.2) are involved in many important facets of steroid hormone activity and metabolism. In this study, Arabidopsis AtST4a and AtST1 were identified and characterized as brassinosteroid sulfotransferases that appear to be involved in different aspects of hormone regulation. The two proteins share 44% identity in amino acid sequence, and belong to different plant sulfotransferase families. AtST4a was specific for biologically active end products of the brassinosteroid pathway. The enzyme sulfated brassinosteroids with diverse side-chain structures, including 24-epibrassinosteroids and the naturally occurring (22R, 23R)-28-homobrassinosteroids. AtST4a belongs to a small subfamily of sulfotransferases having two other members, AtST4b and -c. Among the three recombinant enzymes, only AtST4a was catalytically active with brassinosteroids. Transcript expression of AtST4 subfamily members was largely specific to the root. AtST4b- and -c transcript levels were induced by treatment with trans-zeatin, while AtST4a was repressed under the same conditions, supporting a divergent function of AtST4a. Co-regulation of AtST4b and -c correlated with their location in tandem on chromosome 1. AtST1 was stereospecific for 24-epibrassinosteroids, with a substrate preference for the metabolic precursor 24-epicathasterone, and exhibited catalytic activity with hydroxysteroids and estrogens. To gain more insight into this dual activity with plant and mammalian steroids, enzymatic activities of human steroid sulfotransferases toward brassinosteroids were characterized. The dehydroepiandrosterone sulfotransferase SULT2A1 displayed catalytic activity with a selected set of 24-epibrassinolide precursors, including 24-epicathasterone, with specific activities comparable to that measured for the endogenous substrate dehydroepiandrosterone. The comparable activity profiles of AtST1 and SULT2A1 suggest a similar architecture of the acceptor-binding site between the two enzymes, and may potentially reflect a common ability to conjugate certain xenobiotics.

Cloning and Regulation of Flavonol 3-Sulfotransferase in Cell-Suspension Cultures of Flaveria bidentis

Flaveria spp. accumulate flavonol sulfate esters whose biosynthesis is catalyzed by a number of position-specific flavonol sulfotransferases. Although the accumulation of sulfated flavonols appears to be tissue specific and developmentally regulated and to vary among related species, little is known about the mechanism of regulation controlling the synthesis of these metabolites. In the present work, we report the isolation of a cDNA clone from Flaveria bidentis (pBFST3) encoding flavonol 3-sulfotransferase (F3-ST), which catalyzes the first step in the biosynthesis of flavonol poly-sulfates. This clone (pBFST3) was expressed in Escherichia coli and produced an F3-ST with high affinity for the flavonol aglycones, quercetin, and its 7-methyl derivative, rhamnetin. In addition, the synthetic auxin 2,4-dichlorophenoxyacetic acid was shown to induce F3-ST enzyme activity and F3-ST mRNA transcript levels in cell cultures of F. bidentis. The F3-ST mRNA levels increased within the first 3 h, reaching a maximum after 24 h of treatment, and remained elevated for up to 48 h. Treatments with either quercetin 3-sulfate or quercetin 3,7,4[prime]-trisulfate reduced F3-ST enzyme activity in cell cultures but had no effect on the transcript levels. These results are discussed in relation to the putative role of flavonoid conjugates in the regulation of auxin transport.

Chapter Fourteen – Plant Soluble Sulfotransferases: Structural and Functional Similarity with Mammalian Enzymes

This chapter presents the knowledge of plant soluble sulfotransferases (STs) that have been characterized at the molecular level, with an emphasis on their proposed biological function and on the structural determinants involved in substrate recognition and catalysis. The evolutionary link among soluble STs from different phyla is obvious in their common structural domains and the similarity in the chemical structure of their substrates. Recent studies on plant soluble STs have benefited from the Arabidopsis genome project. It is now possible to undertake a systematic characterization of the biochemical and biological functions of all soluble STs present in a higher plant. Future progress in the field will depend on the identification of the unknown metabolites, through the development of methods of extraction and purification compatible with the retention of the labile sulfate group.

Enzymatic synthesis of sulphated flavonols in Flaveria

Abstract Cell-free extracts of Flaveria bidentis and F. chloraefolia catalysed the transfer of sulphate groups from 3′-phosphoadenosine-5′-phosphosulphate to the hydroxyl groups of a variety of hydroxylated and O -methylated flavonols, but not to flavones or phenylpropanoids. Enzymatic sulphation was more predominant at the 3-hydroxyl group, but not to the exclusion of other hydroxyl substituents on the flavonoid ring. Quercetin was sulphated to yield its mono-, di-, tri- and tetrasulphate esters. This, together with the differences observed in the sulphation of different flavonols by extracts of both Flaveria species, suggests the existence of a number of distinct, position-specific sulphotransferases (EC 2.8.2.-). The sulphation reaction was catalysed at an optimum pH of 7.5 in Tris-HCl buffer, required SH groups for activity and was stimulated in the presence of divalent cations.

Computational simulation of a gene regulatory network implementing an extendable synchronous single-input delay flip-flop

We present a detailed and extendable design of the first synchronous single-input delay flip-flop implemented as a gene regulatory network in Escherichia coli (E. coli). The device, which we call the BioD, has one data input (transacting RNA), one clock input (far-red light) and an output that reports the state of the device using green fluorescent protein (GFP). The proposed design builds on Gardners toggle switch, to provide a more sophisticated device that can be synchronized with other devices within the same cell, and which requires only one data input. We provide a mathematical model of the system and simulation results. The results show that the device behaves in line with desired functionality. Further, we discuss the constraints of the design, which pertain to ranges of parameter values. The BioD is extended via the addition of an update function and input and output interfaces. The result is the BioFSM, which constitutes a synchronous and modular finite state machine, which uses an update function to change its state, stored in the BioD. The BioFSM uses its input and output interfaces for inter-cellular communications. This opens the door to the design of a circular cellular automata (the BioCell), which is envisioned as a number of communicating E. coli colonies, each made of clones of one BioFSM.

Automated design of hammerhead ribozymes and validation by targeting the PABPN1 gene transcript

We present a new publicly accessible web-service, RiboSoft, which implements a comprehensive hammerhead ribozyme design procedure. It accepts as input a target sequence (and some design parameters) then generates a set of ranked hammerhead ribozymes, which target the input sequence. This paper describes the implemented procedure, which takes into consideration multiple objectives leading to a multi-objective ranking of the computer-generated ribozymes. Many ribozymes were assayed and validated, including four ribozymes targeting the transcript of a disease-causing gene (a mutant version of PABPN1). These four ribozymes were successfully tested in vitro and in vivo, for their ability to cleave the targeted transcript. The wet-lab positive results of the test are presented here demonstrating the real-world potential of both hammerhead ribozymes and RiboSoft. RiboSoft is freely available at the website http://ribosoft.fungalgenomics.ca/ribosoft/.