Volume 8, Issue 7, Pages 1090-1102 (July 2015) Arabidopsis Squalene Epoxidase 3 (SQE3) Complements SQE1 and Is Important for Embryo Development and Bulk Squalene Epoxidase Activity  Sara Laranjeira, Vitor Amorim-Silva, Alicia Esteban, Monserrat Arró, Albert Ferrer, Rui Manuel Tavares, Miguel Angel Botella, Abel Rosado, Herlânder Azevedo  Molecular Plant  Volume 8, Issue 7, Pages 1090-1102 (July 2015) DOI: 10.1016/j.molp.2015.02.007 Copyright © 2015 The Author Terms and Conditions

Figure 1 Phylogenetic Reconstruction of Plant SQEs. (A) The phylogenetic tree depicts the relationship between Arabidopsis thaliana SQEs and homologs from representative plant genomes (Arabidopsis lyrata, Brachypodium distachyon, Carica papaya, Medicago truncatula, Oryza sativa ssp. japonica, Physcomitrella patens, Selaginella moellendorffii, Solanum lycopersicum, Sorghum bicolor, Thellungiella halophila, Volvox carteri). SQE homologous protein sequences were determined using Phytozome (www.phytozome.net) (Goodstein et al., 2012) and PLAZA (bioinformatics.psb.ugent.be/plaza/) (Proost et al., 2009; Van Bel et al., 2012). Phylogenetic analysis was performed using maximum likelihood with bootstrap analysis (1000 trees). The percentage of trees in which the associated taxa clustered together is shown next to the branching sites. The proposed SQE and SQE-like clusters are highlighted with a gray background. Arabidopsis thaliana SQEs are highlighted in bold. Database reference accession/annotation codes for each protein are indicated, as well as tentative nomenclature for future reference (square brackets). (B) Synteny plot of the SQE-like genomic block in Arabidopsis thaliana, Arabidopsis lyrata, Brassica rapa (Nappa cabbage), Capsella rubella (Shepherd’s purse) and Carica papaya, using VISTA (http://genome.lbl.gov/vista/index.shtml) (Frazer et al., 2004). Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 2 Histochemical Analysis of Promoter-Driven GUS Activity in pSQE2::GUS and pSQE3::GUS Plants. (A) Schematic representation of the size and position of the promoters used for both constructs. (B–L) GUS histochemical staining for shoots of 10-day-old seedlings (B), cotyledons of 10-day-old seedlings (C), roots of 10-days-old seedlings (D), root tips of 10-days-old seedlings (E), stomata of 10-day-old seedlings (F), mature leaves of 1-month-old plants (G), shoot inflorescence of 1-month-old plants (H), flowers of 1-month-old plants (I), developing siliques (J), fully matured siliques (K), seeds (L). Scale bars represent 1 cm (G), 1 mm (B, C, H–K), 0.1 mm (D, E), 20 μm (F, L). Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 3 The sqe3-1 Mutant Displays a Terbinafine Sensitivity Phenotype. (A) Schematic representation of squalene epoxidase genes SQE1, SQE2, and SQE3. The positions of gene-specific PCR primers used for diagnostic PCR genotyping of the T-DNA insertion (SQE# dPCR LP and RP) and RT–PCR analysis (SQE# RTFw and Rv) are represented by arrows. Exons are represented by dark boxes, introns by dark lines, and the T-DNA insertion by a triangle. Upstream of the ATG is the indication of the estimated promoter size as predicted by the Arabidopsis Gene Regulatory Information Server (AGRIS) database (Davuluri et al., 2003). (B) RT–PCR analysis of sqe2-1 and sqe3-1 single and sqe2-1/sqe3-1 double mutants, with background Col-0 as a control. ACT2 was used as a control gene with constitutive expression. (C) Morphology of 7-day-old mutant seedlings (sqe2-1, sqe3-1 and sqe2-1/sqe3-1). Scale bar represents 0.5 mm. (D) Morphology of 1-month-old sqe2-1, sqe3-1, and sqe2-1/sqe3-1 mutants. Scale bar represents 0.5 cm. (E) Terbinafine (Tb) sensitivity phenotype assay; plants developing green leaves were considered resistant. Scale bar represents 1 cm. (F) Quantification of seedling viability of Col-0, sqe2-1, and sqe3-1 was evaluated in vitro in the presence of different concentrations of terbinafine. IC50 represented by dashed lines. Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 4 Analysis of the Profile of Squalene and Several Sterols in Col-0 and sqe3-1 Genotypes. (A) Content of squalene in 14-day-old shoots and roots. (B) Content of sterols in 14-day-old shoots. (C) Content of sterols in 14-day-old roots. Error bars depict means ± SEM (N = 3 for all except Col-0 in (B) for which N = 2). Indications represent significantly different levels of the mutant in relation to the control (unpaired t-test with 95% confidence interval; n.s., nonsignificant; *P < 0.05). Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 5 Suppression of sqe1-5 Shoot (Scion) Defects by sqe3-1 Rootstocks. (A) sqe1-5 shoot (scion) recovers the wild-type phenotype when grafted onto sqe3-1 rootstock. Self-grafted sqe1-5/sqe1-5 and sqe3-1/sqe3-1 were used as controls. A total of 15 viable plants per graft combination were analyzed and a representative plant per combination is depicted after 15 days of growth in soil. Scale bar represents 1 cm. (B) Diagnostic PCR validation of scion and rootstock genotypes. MW, molecular weight marker. Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 6 Analysis of SQE1 and SQE3 Subcellular Localization and Functional Complementation of the sqe1 Allele by SQE3 Expression. (A and B) Developmental phenotypes of SQE1-GFP and SQE3-GFP overexpression in the sqe1-5 mutant background, in both in vitro 2-weeks-old seedlings (A) and soil-grown 3-week-old plants (B). Scale bar represents 1 cm. (C–H) Confocal images showing co-localization of SQE1-GFP (C–E) and SQE3-GFP (F–H) green fluorescence with ER-Tracker Red fluorescence, in Arabidopsis thaliana. Scale bar represents 10 μm. Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions

Figure 7 The sqe1-5/sqe1-5 sqe3-1/sqe3-1 Double Mutant Is Infertile. (A) Morphology of plants genotyped for the isolation of sqe1-5/sqe3-1 double mutants at 1-month-old stage from a sqe1-5/sqe1-5 SQE3/sqe3-1 parent; inset, adult sqe1-5/sqe1-5 sqe3-1/sqe3-1 plant. (B) Embryo lethal phenotype of the sqe1-5/sqe1-5 sqe3-1/sqe3-1 progeny. (C) Siliques derived from sqe1-5/sqe1-5 SQE3/sqe3-1 and sqe1-5/sqe1-5 SQE3/SQE3 genotypes. White triangles represent aborted seeds. (D, E) Seed analysis on the sqe1-5/sqe1-5 SQE3/sqe3-1 and sqe1-5/sqe1-5 SQE3/SQE3 genotypes. (D) Total number of seeds per silique. (E) Seed abortion rate. Error bars represent SEM (N ≥ 30). Asterisks represent significantly different levels between genotypes (Student t-test; *P < 0.05; ***P < 0.001). Scale bar represents 1 cm (A) and 1 mm (B, C). Molecular Plant 2015 8, 1090-1102DOI: (10.1016/j.molp.2015.02.007) Copyright © 2015 The Author Terms and Conditions