Figure 8: AUX1 over-expressing lines and snl1snl2 mutant show increased cell proliferation and CYCDs expression in the radicle during germination.

(a) Cell numbers in the radicle of 24 h imbibed seeds from Col, 12Spro::AUX1 and snl1snl2 were counted after FM4-64 (T-3166; Molecular Probes) staining and fluorescence microscopy imaging. The images in the left show stained radicles. The graphs in the right show endodermis cell numbers in Col, 12Spro::AUX1 and snl1snl2 with s.d. (n>30). This experiment was performed two times with independent plants, similar results were obtained and a representative result is shown. Scale bars, 50 μm. (b) Changes in nuclear ploidy levels during seed imbibition. Histograms representing DNA content of embryo cells during seed imbibition. After-ripened seeds of Col, 12Spro::AUX1 and snl1snl2 were imbibed for 24 h (upper panel) or 36 h (lower panel). 2C peak, G1 DNA content; 4C peak, G2 DNA content; 8C and 16C peaks, endoreduplicating cells. The graphs in the right show a quantification of the cell numbers at the different stages of the cell cycle based on the histograms. The bars indicate s.d. of three biological replicates and different letters indicate a significant difference determined by Tukey’s honestly significant difference (HSD) test (P<0.05). (c) The relative expression of CYCD1;1 and CYCD4;1 in imbibed fully after-ripened seeds from Col and snl single and double mutants measured by qRT–PCR. Transcript levels were normalized to the level of ACTIN8. The relative transcript level in wild-type Col is set as one. The bars indicate s.e.’s of three biological replicates. (d) The relative expression of AUX1, CYCD1;1 and CYCD4;1 in fully after-ripened dry and imbibed seeds from Col and three 12Spro::AUX1 transgenic lines measured by qRT–PCR. Transcript levels were normalized to the level of ACTIN8. The bars indicate s.e.’s of three biological replicates. (e) Germination phenotypes of 12Spro::AUX1/cycd4;1 (top) and the triple mutant cycd4;1snl1snl2-1 (bottom). Seeds were stratified by imbibition for 3 days at 4 °C before germination. Percentages of seed germination are means (±s.d.) based on the seeds from eight individual plants. Different letters at the same timepoint indicate a significant difference determined by Tukey’s HSD test (P<0.05).