Functional identification of sterol‐4α‐methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol‐4α‐methyl oxidation¹

Abstract

Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4K K-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms

“…In any case, modification of the sterol composition observed in smo2-1/+ smo2-2 and smo2-1 smo2-2/+ mutants is probably responsible for the observed phenotype, because the proper membrane sterol composition has already been shown to be essential for correct PIN auxin efflux facilitator positioning and auxin-dependent developmental processes (Souter et al, 2002;Willemsen et al, 2003;Men et al, 2008;Pullen et al, 2010). SMO2-1 and SMO2-2 share 91% amino acid sequence identity, and both can rescue the yeast erg25 mutant (Darnet et al, 2001;Darnet and Rahier, 2004), suggesting that they exert similar functions in yeast. Consistently, we observed no obvious phenotype in single homozygous smo2-1 or smo2-2 mutants, but the smo2-1 smo2-2 double mutant was embryonic lethal, indicating that SMO2-1 and SMO2-2 share essential but redundant functions in Arabidopsis.…”

“…Although previous studies clearly revealed that SMO2s function biochemically as 4a-methyl-Δ 7 -sterol4a-methyl oxidases (Darnet et al, 2001;Darnet and Rahier, 2004), the physiological functions of SMO2s remain elusive. Silencing of SMO2s in Nicotiana benthamiana using the VIGS approach profoundly altered the sterol profiles of the infected plants but caused no phenotype (Darnet and Rahier, 2004).…”

“…However, in higher plants, the two C-4 demethylation reactions are interrupted by several other steps (Supplemental Fig. S1; Rahier, 2011), and two distinct families of SMO enzymes (SMO1 and SMO2, containing three and two members, respectively) are involved in the first and second C-4 demethylation steps (Darnet et al, 2001;Darnet and Rahier, 2004). Members of the SMO1 and SMO2 protein families share 32% and 29% sequence identity, respectively, with the yeast (Saccharomyces cerevisiae) homolog ERG25, but only SMO2 family proteins can complement the yeast erg25 mutant (Darnet et al, 2001;Darnet and Rahier, 2004).…”

“…S1; Rahier, 2011), and two distinct families of SMO enzymes (SMO1 and SMO2, containing three and two members, respectively) are involved in the first and second C-4 demethylation steps (Darnet et al, 2001;Darnet and Rahier, 2004). Members of the SMO1 and SMO2 protein families share 32% and 29% sequence identity, respectively, with the yeast (Saccharomyces cerevisiae) homolog ERG25, but only SMO2 family proteins can complement the yeast erg25 mutant (Darnet et al, 2001;Darnet and Rahier, 2004). Virus-induced gene silencing (VIGS) in tobacco showed that, after the silencing of SMO1, 4,4-dimethyl-9b,19-cyclopropylsterols were accumulated, whereas after the silencing of SMO2, 4a-methyl-D 7 -sterols were accumulated (Darnet and Rahier, 2004;Rahier, 2011).…”

5 Sterol metabolism and functions in higher plants

“…2). The enzymatic characterization of these complex systems is actually under investigation (Pascal et al 1994;Darnet et al 2001). Opening of the cyclopropane ring of cycloeucalenol by COI has been thoroughly studied (Rahier et al 1989); it is a major target for morpholine fungicides, which trigger accumulation of β,19-cyclopropylsterols (Benveniste and Rahier 1992).…”