Global poly(A) site analysis of the four genotypes studied in this report.

<p>Poly(A) site distributions in extended 3′-UTRs were determined on a gene-by-gene basis and used for all possible pair-wise comparisons (wt-<i>oxt6</i>, wt-C30G, etc.) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#s2" target="_blank">Methods</a>. Cumulative plots of the difference metric for each pairwise comparison were generated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Thomas1" target="_blank">[25]</a> and are shown here. A. Plot of data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Thomas1" target="_blank">[25]</a>, showing the results of comparisons for replicates from the same line (in this case, the wt; “wt-wt leaf”) the wt and <i>oxt6</i> mutant (“wt-oxt6 leaf”). These curves represent the expected “extremes” of similarity and differences, respectively. B. The comparisons involving the wt, C30G, and G30GM lines were superimposed on those shown in panel A. C. The three comparisons of the <i>oxt6</i> mutant with the other lines were superimposed on those shown in panel A.</p

Structures of the transgenes assembled for this study, with an insert showing the wt (A) and mutant CAM-binding domain (B).

<p>A, the genomic DNA is shown at the top, and the two mRNAs beneath. Dark gray boxes indicate the exons present in <i>CPSF30</i>, the smaller of the two transcripts of the <i>OXT6</i> gene; light boxes are additional exons in <i>CPSF30-YT521B</i>, the larger of the two transcripts of <i>OXT6</i>. For brevity, and because the structures are identical for the mutation illustrated at the bottom, only the genomic DNA of the wild-type transgene is shown. The sequences at the bottom of B show the changes used to create the calmodulin-binding mutant; these have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Delaney1" target="_blank">[18]</a>.</p

Characteristics of o<i>xt6</i> flowers.

<p>(A) A wild-type inflorescence showing selected stages of flower development. (B) and (C) flowers from wild-type and oxt6 plants at the +1 stage and +3 stage, respectively. (D) Lengths of the stamens at +1 stage, calculated as a fraction of the lengths of pistils (a value of 1.0 indicates that both organs are the same length). The difference between the wild-type and mutant was significant ate the p<0.05 level by the Student's t-test. (E) Silique phenotypes of the wild-type and <i>oxt6</i> mutant. A typical wild-type inflorescence is shown on the left, and an <i>oxt6</i> inflorescence on the right. The inflorescence in the middle is also from an <i>oxt6</i> plant; in this infloresence, the flowers indicated by the dark arrows were hand-pollinated with pollen from the same <i>oxt6</i> plant.</p

Lateral root development of the <i>oxt6</i> mutant and complemented plants.

<p>(A) Image of 10-days-old wild-type (wild-type, left) and <i>oxt6</i> (right) seedlings grown on a half MS medium plate. (B) Numbers of lateral roots per primary root per cm in wild-type, <i>oxt6, oxt6</i>::C30G, and <i>oxt6</i>::C30GM lines. Plants were germinated and grown on vertical plates for 10 days, when lateral roots were counted. (C) Primordia in the wild-type and <i>oxt6</i> mutant. Primordia were counted on whole-mount roots of wild-type and <i>oxt6</i> plants 10 days after germination and growth on vertical plates. (D) Numbers of primordia at the different stages of lateral root development (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Malamy1" target="_blank">[23]</a>). <i>n</i>>8 plants per column.</p

Results of an analysis of global gene expression in the roots of the wt and <i>oxt6</i> mutant.

<p>Gene expression was measured using the RNA-Seq functionality of CLC Genomics Workbench, and the results analyzed in MAPMAN as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#s2" target="_blank">Methods</a>. Bins representing functional groups of genes whose expression is significantly different in the wt and mutant were identified and the p-values that describe the conformance with the hypothesis that the two backgrounds are identical were plotted as shown; for this, the log(10) values for the reciprocal of each p-value was calculated and used in the graph. Bins mentioned in the text are color-coded and described beneath the plot. The full set of bins and p-values for this analysis is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779.s002" target="_blank">S2 Table</a>.</p