Limits...
SDG2-mediated H3K4 methylation is required for proper Arabidopsis root growth and development.

Yao X, Feng H, Yu Y, Dong A, Shen WH - PLoS ONE (2013)

Bottom Line: Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre.Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance.We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, School of Life Sciences, Fudan University, Shanghai, PR China.

ABSTRACT
Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

Show MeSH

Related in: MedlinePlus

Loss of SDG2 partially affects auxin regulation in roots. Aand 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585709&req=5

pone-0056537-g004: Loss of SDG2 partially affects auxin regulation in roots. Aand 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.

Mentions: To gain further insight into the mechanisms underlying the sdg2-3 root SCN defect, we introgressed into sdg2-3 the marker DR5:GFP which reports auxin signaling in single cells [45], [46]. In WT roots expressing DR5:GFP, the GFP signal was detected at high levels in columella cells, columella initial cells and QC cells (Figure 4A). In sdg2-3 roots, the intensity of GFP signal appeared slightly weaker and most importantly the auxin gradient and maximum in QC were lost; almost no GFP signal could be detected at QC position (Figure 4B). Next, we performed quantitative real-time RT-PCR analysis for auxin-related genes to compare their expression in WT and sdg2-3 roots. As shown in Figure 4C, expression of each IAA14, IAA19, IAA29, IAA34, and to a lesser extent of IAA28, was significantly increased whereas expression of IAA2, IAA16 and IAA30 was unchanged in sdg2-3. An increase of expression in sdg2-3 was also observed for the cell cycle inhibitory gene RETINOBLASTOMA RELATED (RBR, Figure 4C). Because RBR and several IAA genes are known to negatively regulate root growth [9], [14], [17], [47], their upregulation is consistent with the root growth suppression phenotype of the sdg2-3 mutant. Nevertheless, because SDG2 acts as an activator of gene transcription [39], [40], it is likely that the observed gene upregulation is caused indirectly by the sdg2-3 mutation. An auxin-mediated PLETHORA (PLT) pathway is essential for root SCN maintenance [48], consistently the expression of PLT1 (but not PLT2) was reduced in sdg2-3 roots (Figure 4C).


SDG2-mediated H3K4 methylation is required for proper Arabidopsis root growth and development.

Yao X, Feng H, Yu Y, Dong A, Shen WH - PLoS ONE (2013)

Loss of SDG2 partially affects auxin regulation in roots. Aand 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585709&req=5

pone-0056537-g004: Loss of SDG2 partially affects auxin regulation in roots. Aand 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.
Mentions: To gain further insight into the mechanisms underlying the sdg2-3 root SCN defect, we introgressed into sdg2-3 the marker DR5:GFP which reports auxin signaling in single cells [45], [46]. In WT roots expressing DR5:GFP, the GFP signal was detected at high levels in columella cells, columella initial cells and QC cells (Figure 4A). In sdg2-3 roots, the intensity of GFP signal appeared slightly weaker and most importantly the auxin gradient and maximum in QC were lost; almost no GFP signal could be detected at QC position (Figure 4B). Next, we performed quantitative real-time RT-PCR analysis for auxin-related genes to compare their expression in WT and sdg2-3 roots. As shown in Figure 4C, expression of each IAA14, IAA19, IAA29, IAA34, and to a lesser extent of IAA28, was significantly increased whereas expression of IAA2, IAA16 and IAA30 was unchanged in sdg2-3. An increase of expression in sdg2-3 was also observed for the cell cycle inhibitory gene RETINOBLASTOMA RELATED (RBR, Figure 4C). Because RBR and several IAA genes are known to negatively regulate root growth [9], [14], [17], [47], their upregulation is consistent with the root growth suppression phenotype of the sdg2-3 mutant. Nevertheless, because SDG2 acts as an activator of gene transcription [39], [40], it is likely that the observed gene upregulation is caused indirectly by the sdg2-3 mutation. An auxin-mediated PLETHORA (PLT) pathway is essential for root SCN maintenance [48], consistently the expression of PLT1 (but not PLT2) was reduced in sdg2-3 roots (Figure 4C).

Bottom Line: Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre.Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance.We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, School of Life Sciences, Fudan University, Shanghai, PR China.

ABSTRACT
Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

Show MeSH
Related in: MedlinePlus