Volume 5, Issue 6, Pages 1295-1309 (November 2012) XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms, Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature  Sofia D. Carvalho, Rita Saraiva, Teresa M. Maia, Isabel A. Abreu, Paula Duque  Molecular Plant  Volume 5, Issue 6, Pages 1295-1309 (November 2012) DOI: 10.1093/mp/sss048 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Alternative Splicing of the XBAT35 Gene. Schematic representation of the genomic structure of the XBAT35 gene (boxes indicate exons with UTRs in gray and lines between boxes represent introns), the coding sequence of the two splice variants, XBAT35.1 and XBAT35.2, generated by alternative splicing (the skipped exon is shown in black), and the two corresponding predicted protein isoforms. The inset shows RT–PCR amplification of the two XBAT35 transcripts using primers F1 and R1, whose location is indicated by the arrows in the genomic scheme. ANK, ankyrin repeat; NLS, nuclear localization signal; RING, Really Interesting New Gene domain. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Expression Patterns of the XBAT35 Gene. (A) RT–PCR analysis of XBAT35 transcript levels in whole seedlings and different tissues from wild-type (Col-0) plants. (B) XBAT35 expression levels in rosette leaves of 5-week-old plants grown under long-day (LD) or short-day (SD) photoperiod regimes, represented by the diagrams on the right (white and black boxes indicate light and dark cycles, respectively, while dashed lines indicate the time points at which the plant material was harvested). (C) Expression of XBAT35 in 2-week-old seedlings exposed to cold (4°C, 36 h) or heat (37°C, 3 h) stress, NaCl (250 mM, 6 h), glucose (6%, 9 h), ABA (3 µM, 48 h), IAA (10 µM, 6 h), ACC (10 µM, 6 h), or AgNO3 (100 µM, 6 h). For each condition, treatment effectiveness was confirmed using the indicated positive control gene. The cyclophilin (ROC1) and the actin2 (ACT2) genes are shown as loading controls. Results are representative of at least three independent experiments. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Subcellular Localization of the Two XBAT35 Splice Isoforms. Representative images of onion epidermal cells (A) or isolated Arabidopsis protoplasts (B) transiently transformed with YFP fusions of XBAT35.1 and XBAT35.2 under the control of the 35S promoter. YFP alone was used as a control (A, B) and DAPI as a nuclear marker (B). Scale bars = 50 µm. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 In Vitro E3 Ubiquitin Ligase Activity of the Two XBAT35 Isoforms. His-tagged recombinant versions of XBAT35.1 and XBAT35.2 were incubated with His-tagged ubiquitin in the presence or absence of rabbit E1, human E2 UbcH5b, or ATP for 2 h at 30°C. Samples were resolved by 10% SDS–PAGE and XBAT35 proteins detected by Western blot analysis using anti-His antibody. The blot on the left shows each isoform run alone. The molecular mass (in kDa) of protein markers is shown on the left of the blots. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Isolation of XBAT35 Loss-of-Function Lines. (A) Schematic representation of the XBAT35 gene showing the site of insertion and orientation of the T-DNA in xbat35-1 and xbat35-2 insertion lines (boxes indicate exons with UTRs in gray and the alternatively spliced exon in black, lines between boxes represent introns, and arrows indicate the location of XBAT35- and T-DNA-specific primers used in genotyping and mRNA analysis). (B) PCR-based genotyping of homozygous xbat35-1 and xbat35-2 lines. (C) RT–PCR analysis of XBAT35 transcript levels in xbat35-1 and xbat35-2 seedlings. The ubiquitin10 (UBQ10) and cyclophilin (ROC1) genes were used as loading controls. (D) RT–PCR analysis of XBAT35 transcript levels in two independent XBAT35 RNAi silencing lines and the xbat35-1 insertion mutant. Cyclophilin (ROC1) is shown as a loading control. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Ethylene Phenotype of XBAT35 Loss-of-Function Lines. (A) Hypocotyl length of Col-0, xbat35-1, two independent XBAT35-RNAi lines, Ws and xbat35-2 seedlings grown 3 d in the dark under control conditions (white bars) or in the presence of 10 µM ACC (gray bars) or 25 µM ACC (black bars) supplemented or not with 100 µM AgNO3 (means ± SE, n = 40–60). (B) Apical hook curvature of Col-0, xbat35-1, two independent XBAT35-RNAi lines, Ws and xbat35-2 seedlings grown 3 d in the dark under control conditions (white bars) or in the presence of 10 µM ACC (gray bars) or 10 µM ACC supplemented with 100 µM AgNO3 (black bars). The ctr1-1, ein3-1, and hls1-1 hook mutants were included as controls. The inset illustrates the determination of the hook angle (means ± SE, n = 40–60). Asterisks indicate significantly different values (* P < 0.05; ** P < 0.01; *** P < 0.001) from the corresponding wild-type according to Student’s t-test. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Complementation of the xbat35-1 Ethylene-Mediated Apical Hook Curvature Phenotype. (A) RT–PCR analysis of XBAT35 expression in light-grown seedlings of the wild-type (Col-0), mutant (xbat35-1) and six independent complementation lines expressing either the XBAT35.1 (cXBAT35.1a-c) or XBAT35.2 (cXBAT35.2a-c) splice variants. Ubiquitin10 (UBQ10) is shown as a loading control. (B) Apical hook curvature of Col-0, xbat35-1, and the six complementation lines grown for 3 d in the dark under control conditions (white bars) or in the presence of 10 µM (black bars) ACC (means ± SE, n = 40–60). Asterisks indicate significantly different values (* P < 0.05; ** P < 0.01; *** P < 0.001) from the wild-type according to Student’s t-test. (C) Representative images of Col-0, xbat35-1, cXBAT35.1a, and cXBAT35.2a seedlings grown in the dark under control conditions or in the presence of 10 µM ACC. Scale bar = 1 mm. Molecular Plant 2012 5, 1295-1309DOI: (10.1093/mp/sss048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions