Transparent testa16 plays multiple roles in plant development and is involved in lipid synthesis and embryo development in canola

Abstract

Transparent Testa16 (TT16), a transcript regulator belonging to the B(sister) MADS box proteins, regulates proper endothelial differentiation and proanthocyanidin accumulation in the seed coat. Our understanding of its other physiological roles, however, is limited. In this study, the physiological and developmental roles of TT16 in an important oil crop, canola (Brassica napus), were dissected by a loss-of-function approach. RNA interference (RNAi)-mediated down-regulation of tt16 in canola caused dwarf phenotypes with a decrease in the number of inflorescences, flowers, siliques, and seeds. Fluorescence microscopy revealed that tt16 deficiency affects pollen tube guidance, resulting in reduced fertility and negatively impacting embryo and seed development. Moreover, Bntt16 RNAi plants had reduced oil content and altered fatty acid composition. Transmission electron microscopy showed that the seeds of the RNAi plants had fewer oil bodies than the nontransgenic plants. In addition, tt16 RNAi transgenic lines were more sensitive to auxin. Further analysis by microarray showed that tt16 down-regulation alters the expression of genes involved in gynoecium and embryo development, lipid metabolism, auxin transport, and signal transduction. The broad regulatory function of TT16 at the transcriptional level may explain the altered phenotypes observed in the transgenic lines. Overall, the results uncovered important biological roles of TT16 in plant development, especially in fatty acid synthesis and embryo development.

Figure 1.

RNAi-mediated silencing of Bntt16 expression. A, Gene expression patterns (2^−ΔCT) of BnTT16s in canola tissues. BnTT16 expression levels in the stamen are also shown in the inset. B, Overall expression levels of BnTT16s in samples 2 DAF were down-regulated in all RNAi lines. Bntt16 expression level in NT plants was set as 1 for comparison. 4-1, tt16 RNAi transgenic line 4-1.

Figure 2.

Morphological alteration exhibited by the tt16 down-regulated canola line. A, Dwarf phenotypes of 40-d-old tt16 RNAi transgenic lines. B, Leaves of 40-d-old transgenic lines. C, Delayed flowers of 70-d-old tt16 RNAi transgenic lines. D, Flowers of 70-d-old transgenic lines. E, Number of flowers and inflorescences in 70-d-old tt16 RNAi transgenic lines. 4-1, tt16 RNAi transgenic line 4-1. sd (n = 3) is indicated by vertical bars. Single and double asterisks indicate significant differences between transgenic and NT plants with P < 0.05 and P < 0.01, respectively, as determined by t test.

Figure 3.

Pollen tubes development in a tt16 RNAi transgenic line and artificial pollination. A, Style section of a NT plants. The arrow indicates a pollen tube. B, Style section of the tt16 RNAi transgenic line 4-1. Bars = 8 μm. C, Number of pollen tubes in style sections. se values (n = 3) are indicated by vertical bars. D, Artificial pollination. 1, NT plants without artificial pollination. 2, NT plants with artificial pollination (self-pollination). 3, NT plants pollinated with line 4-1 (cross-pollination). 4, Line 4-1 without artificial pollination. 5, Line 4-1 with artificial pollination (self-pollination). 6, Line 4-1 pollinated with NT plants (cross-pollination).

Figure 4.

Guiding of pollen tubes to ovules in self- and cross-pollinated plants. A, Self-pollinated NT plants. B, NT plants pollinated with tt16 RNAi transgenic line 4-1. C, Self-pollinated line 4-1. D, Line 4-1 pollinated with NT plants. Arrows indicate pollen targeted into ovules.

Figure 5.

Embryo and seed development in tt16s RNAi transgenic lines. A, Embryo development in line 4-1. B, Mature seeds (43 DAF) of transgenic lines. The single asterisk indicates a type 1 seed with no embryo. The rest are type 2 seeds. C, Embryos in mature seeds (43 DAF). D, Seed transverse sections (34 DAF) of NT plants. E, Seed transverse sections (34 DAF) of line 4-1. Co, Cotyledon; Hy, hypocotyl. Bar = 40 mm.

Figure 6.

Transmission electron microscopy observation of hypocotyl cells (34 DAF) in tt16 RNAi transgenic lines. A, Transmission electron microscopy observation of hypocotyl cells. Ob, Oil bodies. Bars = 2.5 μm. B, Quantitation of the area occupied by oil bodies. The percentage area occupied by oil bodies was determined using ImageJ software (http://rsbweb.nih.gov/ij/). se values (n = 3) are indicated by vertical bars. The single asterisk indicates a significant difference between transgenic and NT plants with P < 0.05 as determined by t test. 4-1, tt16 RNAi transgenic line 4-1.

Figure 7.

Verification of microarray data using real-time PCR. A, Genes involved in lipid metabolism. B, Genes involved in gynoecium and embryo development. C, Auxin-related genes.

Figure 8.

Auxin dose response of tt16 RNAi transgenic lines. Hypocotyl explants of 9-d-old canola seedlings were incubated on one-half-strength Murashige and Skoog medium containing the indicated naphthaleneacetic acid concentrations for 20 d. 4-1, tt16 RNAi transgenic line 4-1.