Hubert Schaller

The role of sterols in plant growth and development.

Sterols found in all eukaryotic organisms are membrane components which regulate the fluidity and the permeability of phospholipid bilayers. Certain sterols in minute amounts, such as campesterol in Arabidopsis thaliana, are precursors of oxidized steroids acting as growth hormones collectively named brassinosteroids. The crucial importance of brassinosteroids upon growth and development has been established through the study of a set of dwarf mutants affected in brassinosteroid synthesis or perception. Some of these dwarfs are, in fact, deficient in the final steps of sterol biosynthesis and their developmental phenotypes are primarily caused by a depletion in the sterol precursor for brassinosteroids. Recently, the characterization of genes encoding sterol biosynthetic enzymes and the isolation of novel plant lines affected in the expression of those genes, either by insertional or classical mutagenesis, overexpression or cosuppression, have shed new light on the involvement of sterols in biological processes such as embryonic development, cell and plant growth, and fertility, which will be presented and discussed in this review article.

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction.

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473–484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.

A different function for a member of an ancient and highly conserved cytochrome P450 family: From essential sterols to plant defense

CYP51 sterol demethylases are the only cytochrome P450 enzymes with a conserved function across the animal, fungal, and plant kingdoms (in the synthesis of essential sterols). These highly conserved enzymes, which are important targets for cholesterol-lowering drugs, antifungal agents, and herbicides, are regarded as the most ancient member cytochrome P450 family. Here we present a report of a CYP51 enzyme that has acquired a different function. We show that the plant enzyme AsCYP51H10 is dispensable for synthesis of essential sterols and has been recruited for the production of antimicrobial compounds (avenacins) that confer disease resistance in oats. The AsCyp51H10 gene is synonymous with Sad2, a gene that we previously had defined by mutation as being required for avenacin synthesis. In earlier work, we showed that Sad1, the gene encoding the first committed enzyme in the avenacin pathway (β-amyrin synthase), had arisen by duplication and divergence of a cycloartenol synthase-like gene. Together these data indicate an intimate evolutionary connection between the sterol and avenacin pathways. Sad1 and Sad2 lie within 70 kb of each other and are expressed specifically in the epidermal cells of the root tip, the site of accumulation of avenacins. These findings raise intriguing questions about the recruitment, coevolution, and regulation of the components of this specialized defense-related metabolic pathway.

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites.

Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats.

Neurons have a high demand for cholesterol to develop and maintain membrane‐rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum‐free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell‐specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post‐squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol‐converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up‐regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.

Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non–photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3–oxidosqualene–triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS–triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, β– and α–amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUP1 and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.

CYP86B1 Is Required for Very Long Chain ω-Hydroxyacid and α,ω-Dicarboxylic Acid Synthesis in Root and Seed Suberin Polyester

Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid ω-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven β-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22- and C24-hydroxyacids and α,ω-dicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development.

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant.

Abstract. In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression.

Allelic mutant series reveal distinct functions for Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis

Sterols have multiple functions in all eukaryotes. In plants, sterol biosynthesis is initiated by the enzymatic conversion of 2,3-oxidosqualene to cycloartenol. This reaction is catalyzed by cycloartenol synthase 1 (CAS1), which belongs to a family of 13 2,3-oxidosqualene cyclases in Arabidopsis thaliana. To understand the full scope of sterol biological functions in plants, we characterized allelic series of cas1 mutations. Plants carrying the weak mutant allele cas1–1 were viable but developed albino inflorescence shoots because of photooxidation of plastids in stems that contained low amounts of carotenoids and chlorophylls. Consistent with the CAS1 catalyzed reaction, mutant tissues accumulated 2,3-oxidosqualene. This triterpenoid precursor did not increase at the expense of the pathway end products. Two strong mutations, cas1–2 and cas1–3, were not transmissible through the male gametes, suggesting a role for CAS1 in male gametophyte function. To validate these findings, we analyzed a conditional CRE/loxP recombination-dependent cas1–2 mutant allele. The albino phenotype of growing leaf tissues was a typical defect observed shortly after the CRE/loxP-induced onset of CAS1 loss of function. In the induced cas1–2 seedlings, terminal phenotypes included arrest of meristematic activity, followed by necrotic death. Mutant tissues accumulated 2,3-oxidosqualene and contained low amounts of sterols. The vital role of sterols in membrane functioning most probably explains the requirement of CAS1 for plant cell viability. The observed impact of cas1 mutations on a chloroplastic function implies a previously unrecognized role of sterols or triterpenoid metabolites in plastid biogenesis.