Figure 1.
Structures of the transgenes assembled for this study, with an insert showing the wt (A) and mutant CAM-binding domain (B).
A, the genomic DNA is shown at the top, and the two mRNAs beneath. Dark gray boxes indicate the exons present in CPSF30, the smaller of the two transcripts of the OXT6 gene; light boxes are additional exons in CPSF30-YT521B, the larger of the two transcripts of OXT6. 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 [18].
Figure 2.
Root length assay of the response of the oxt6 mutant to methyl viologen.
(A) Methyl violgen treatment. Plants with the indicated genotypes were grown as described in the Methods on media containing 50 nM methyl viologen; root lengths were measured after 10 days of growth. (B) Without methyl violgen treatment. The same growth condition as in A but without added methyl viologen. Wt, the wild-type parent of the oxt6 mutant; oxt6, the oxt6 mutant; C30G-1, the C30G-1 line whose expression is assessed in Figure S2 in S1 File; C30G-2, line C30G2-2 in Figure S2 in File S1; C30GM-1, line C30GM-1 in Figure S2 in S1 File; C30GM-2, line C30GM-2 in Figure S2 in S1 File. Error bars denote standard deviations; * indicates a difference from the wild-type with a p-value <0.05 (Student's t-test, n≥12).
Figure 3.
Lateral root development of the oxt6 mutant and complemented plants.
(A) Image of 10-days-old wild-type (wild-type, left) and oxt6 (right) seedlings grown on a half MS medium plate. (B) Numbers of lateral roots per primary root per cm in wild-type, oxt6, oxt6::C30G, and oxt6::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 oxt6 mutant. Primordia were counted on whole-mount roots of wild-type and oxt6 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 [23]). n>8 plants per column.
Figure 4.
Characteristics of oxt6 flowers.
(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 oxt6 mutant. A typical wild-type inflorescence is shown on the left, and an oxt6 inflorescence on the right. The inflorescence in the middle is also from an oxt6 plant; in this infloresence, the flowers indicated by the dark arrows were hand-pollinated with pollen from the same oxt6 plant.
Figure 5.
Responses of the wild-type, oxt6 mutant, and complemented transgenic lines to a battery of hormones.
The primary root length of wild-type, oxt6, and complemented lines was determined after 10 days of growth on half MS medium with the indicated concentrations of IAA (A), 6-BA (B), GA3 (C); the hypocotyl length on ACC (D). * indicates a statistically significant difference (p<0.05, Student's t-test) from the wild-type.
Figure 6.
Global poly(A) site analysis of the four genotypes studied in this report.
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-oxt6, wt-C30G, etc.) as described in Methods. Cumulative plots of the difference metric for each pairwise comparison were generated as described [25] and are shown here. A. Plot of data from [25], showing the results of comparisons for replicates from the same line (in this case, the wt; “wt-wt leaf”) the wt and oxt6 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 oxt6 mutant with the other lines were superimposed on those shown in panel A.
Figure 7.
Results of an analysis of global gene expression in the roots of the wt and oxt6 mutant.
Gene expression was measured using the RNA-Seq functionality of CLC Genomics Workbench, and the results analyzed in MAPMAN as described in Methods. 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 S2 Table.