Figure 1.
Loss of SDG2 reduces both primary and lateral root growth. A,
Phenotypes of wild-type Col and the mutant sdg2-3 seedlings at 26 days after germination. Bar = 1 cm. B, Comparison of primary root length between Col and sdg2-3 from 4 to 18 days after germination. C, Comparison of lateral root number between Col and sdg2-3 from 8 to 18 days after germination. All data are mean values from two independent experiments with each of at least 20 plants. Bars indicate SD.
Figure 2.
Loss of SDG2 distinctively affects different developmental stages of lateral root formation. A,
Developmental stages of lateral root formation. Images was captured after histochemical GUS staining of roots from 10-day-old Col seedlings expressing DR5:GUS. Developmental stage nomenclature was according to Malamy and Benfey [43]. Bar = 50 µm. B, Relative distribution of developmental stages of lateral root primordia observed in 10-day-old seedlings of the wild-type Col and the mutant sdg2-3. Primordia were counted and examined for developmental stages from at least 20 plants, and the experiments were repeated three times. Mean values of percentage are shown and bars indicate SD.
Figure 3.
Loss of SDG2 impairs the primary root stem cell niche maintenance. A
and B, Comparison of primary root apical meristem sizes between wild-type Col and the mutant sdg2-3, respectively. DIC images were taken on the roots of 6-day-old seedlings. Arrowheads indicate positions of the transition from meristem to elongation zone. Bar = 100µm. C and D, Comparison of QC25:GUS expression and root cap cell layer organization between Col and sdg2-3, respectively. DIC images were taken on GUS- and Lugol-stained root tips of 6-day-old seedlings. Arrowheads indicate the columella initial cell layer. Bar = 20 µm. E and F, Comparison of cell layer organization of root apical meristem between Col and sdg2-3, respectively. Confocal images were taken on PI-stained roots of 6-day-old seedlings. Bar = 50 µm. The close-up regions are shown by color indication of different cell types: QC cell in blue, columella root cap and columella initial cells in rose, lateral root cap cells in sky-blue, epidermal cells and epidermis/lateral root cap initials in red, cortex cells in green, endodermal cells in yellow, cortex/endodermis initials in purple, stele cells and stele initials in gray. G and H, Comparison of cell layer organizations of root apical meristem between Col and sdg2-3, respectively. Confocal images were taken on PI-stained roots of 14-day-old seedlings. Bar = 50 µm. The close-up regions are shown with colorations as described in E and F.
Figure 4.
Loss of SDG2 partially affects auxin regulation in roots. A
and B, Comparison of the expression pattern of DR5:GFP reporter in 5-day-old wild-type Col and the mutant sdg2-3, respectively. Note that auxin gradient maximum in QC visualized by DR5:GFP expression in Col is lost in sdg2-3. Bar = 50 µm. C, Relative gene expression levels determined by quantitative RT-PCR analysis. RNA was prepared from roots of 20-day-old Col or sdg2-3 seedlings. RT-PCR was performed using gene specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Effects of exogenous NAA on root elongation of Col and sdg2-3 seedlings. Seeds were germinated and grown on medium containing the indicated concentration of NAA. Root length is shown as a mean value obtained from three independent experiments with each experiment comprising 20 plants. Bar indicates SD. E, Effects of exogenous NAA on lateral root (LR) formation of Col and sdg2-3 seedlings. LR and primordia were counted using the GUS reporter of 10-day-old Col or sdg2-3 seedlings expressing CYCB1;1:GUS. The total number of LR and primordia was divided by root length to report LR formation ability of individual plants. Mean values obtained from three independent experiments and 20 plants per sample per experiment are shown, and bars indicate SD.
Figure 5.
Loss of SDG2 synergistically enhances growth defects of the CAF1 loss-of-function mutant fas2-4. A
, Representative example of 14-day-old seedling of the wild-type Col, the single mutants sdg2-3 and fas2-4, and the double mutant sdg2-3 fas2-4. Bar = 1 cm. B, Comparison of primary root length between fas2-4 and sdg2-3 fas2-4 on 16-day-old seedlings. Root length is shown as a mean value from two independent experiments with each comprising at least 20 plants. Bar indicates SD. C and D, Comparison of root cap cell organization between fas2-4 and sdg2-3 fas2-4, respectively. DIC images were taken on Lugol-stained root tips of 6-day-old seedlings. Arrowhead in C indicates QC position. Bar = 50 µm. E and F, Comparison of cell layer organizations of root apical meristem between fas2-4 and sdg2-3 fas2-4, respectively. Confocal images were taken on PI-stained roots of 6-day-old seedlings. The QC cell in E is marked in blue. Bar = 50 µm.
Figure 6.
Loss of SDG2 drastically reduces nuclear H3K4me3 levels in root cells.
Whole-mount root immunofluorescence staining was performed using an antibody specifically recognizing H3K4me3. Panels from left to right subsequently show confocal images of DAPI, H3K4me3 and merged signals. Close-up images show regions around the root stem cell niche with the QC cells circled. Bar = 50 µm.
Figure 7.
Loss of SDG2 synergistically enhances the CAF1 loss-of-function mutant fas2-4 in causing genome DNA damage but not telomere shortening.
A, Representative comet images of the wild-type Col and the mutants sdg2-3, fas2-4 and sdg2-3 fas2-4. Note the intact nucleus at the left and comet tail formed by fragmented nuclear DNA to the right on each panel. B, DNA damage levels as measured by the percentage of DNA in the comet tails of nuclei for the wild-type and mutants. The mean value of more than 100 nuclei is shown with a SD bar. C, Relative expression levels of DNA repair genes determined by quantitative RT-PCR analysis. RNA was prepared from 14-day-old Col, sdg2-3, fas2-4 or sdg2-3 fas2-4 seedlings. RT-PCR was performed using gene-specific primers and normalized using ACTIN2 as reference. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with Col value setting as 1. Bars indicate SD. D, Telomere length comparison between wild-type and mutants. Genomic DNA was digested with MseI, and DNA gel blot analysis was performed using a DIG-labeled telomere repeat as the probe. Note that telomeres are shortened to similar degree in fas2-4 and sdg2-3 fas2-4 but not in sdg2-3 as compared to Col.