To generate the sog1-7 and suv2-3 mutations, als3-1 seeds were treated with EMS. M2 seedlings were screened for mutants that suppressed the hypersensitivity of als3-1 to Al in a 0.75 mM AlCl3 gel soaked environment. EMS treated seeds were planted around the periphery of the plates and were grown for 7 days at 20°C with a 24 hr continuous light cycle. After 7 days, seedlings with roots that grew to the bottom of the plate were rescued on PNS media. After 2 weeks, putative mutants were transferred to soil and allowed to self-pollinate. M3 seeds were collected from their M2 mutant parent and were screened using the same system. Two of these lines that were able to restore growth on the AlCl3 media, were then further characterized, and after mutation identification named sog1-7 and suv2-3.o generate sog1-7 without als3-1, sog1-7;als3-1 was crossed with Col-0 wild type. F1 lines were allowed to self-pollinate and F2 lines were screened for wild type ALS3 by PCR. The als3-1 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by restriction enzyme digest with ClaI . The wild type ALS3 contains a ClaI site, while als3-1 mutation has lost this site. Digested amplicons were analyzed by gel electrophoresis,plastic garden container resolving wild type or mutant genotype results in a 2% agarose gel. Lines that were homozygous for ALS3 were then screened for the sog1-7 mutation.
The sog1-7mutation can be followed by the amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. To generate suv2-3 without als3-1, suv2-3;als3-1 was crossed with Col-0 wild type. F1 lines were allowed to self-pollinate and F2 lines were screened for wild type ALS3 by PCR. The als3-1 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by restriction enzyme digest with ClaI . The wild type ALS3 contains a ClaI site, while als3-1 mutation has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 2% agarose gel. Lines that were homozygous for ALS3 were then screened for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation. To generate sog1-7;atr-4, sog1-7 was crossed with atr-4. F1 lines were allowed to self-pollinate and F2 lines were screened for sog1-7 by PCR and restriction enzyme digest.
The sog1-7 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. Lines that were homozygous for sog1-7 were then screened for the atr-4 mutation. The atr-4 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel. To generate suv2-3;atr-4, suv2-3 was crossed with atr-4. F1 lines were allowed to self-pollinate and F2 lines were screened atr-4 by PCR and subsequent restriction enzyme digest. The atr-4 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel. Lines that were homozygous for atr-4 were then screened for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation.
To generate the sog1-7 and suv2-3 lines carrying either the CycB1;1::GUS or QC46 reporters, parent lines of sog1-7 and suv2-3 were crossed and F1 seeds allowed to self-pollinate. Genomic DNA was extracted from F2 plants for genotyping. For sog1-7; CyclinB1;1 and sog1-7;QC46 plants were screened to select for plants homozygous for the sog1-7 by PCR and restriction enzyme digest. The sog1-7 mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with DdeI . The wild type SOG1 contains a DdeI site, while sog1-7 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant genotype results in a 3% agarose gel. Plants that were identified as sog1-7 homozygotes were GUS stained to identify lines that were homozygous for the reporter line, either CycB1;1 or QC46. For suv2-3;CyclinB1;1 and suv2-3QC46 plants were first screened to select for GUS activity to identify lines that contained the reporter constructs. Plants that stained positive for GUS were then screened to select for plants homozygous for the suv2-3 mutation. The suv2-3 mutation did not result in the loss of a restriction enzyme recognition site, therefore this mutation was followed by the amplification with markers as listed in Table 1 followed by sequence comparisons to the SUV2 published gene sequence of the Arabidopsis thaliana genome archived on The Arabidopsis Information Resource to identify the suv2-3 mutation.To generate an atr-4 mutant line carrying either the SOG1:GUS or SOG1:GFP reporters, parent lines of atr-4 were crossed to parents of the respective reporter and F1 seeds allowed to self-pollinate. Genomic DNA was extracted from F2 plants.For atr-4;SOG1:GUS the atr-4 mutation was first screened for to select atr-4 homozygous mutants. This mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis,plastic pot resolving wild type or mutant results in a 2% agarose gel. Plants that were identified as atr-4 homozygotes were allowed to mature and seeds from the F3 were harvested for GUS staining to identify lines that were homozygous for the reporter line SOG1:GUS. 30 seeds from the F3 were planted on PNS and stained again for GUS activity. Lines with staining in all 30 seedlings proved to be homozygous for and SOG1:GUS. For atr-4;SOG1:GFP F2 plants were grown on PNS medium containing kanamycin for 10 days and seedlings were selected for resistance to the antibiotic. Selected individuals were allowed to recover on PNS and transplanted into soil until maturity. The F3 seeds were then harvested, and 30 seeds were planted again on PNS media containing kanamycin, and lines with 100% germination proved to be homozygous for kanamycin resistance, and therefore SOG1:GFP homozygotes.
Plants that were identified as SOG1:GFP homozygotes were then screened for the atr-4 mutation. This mutation can be followed by amplification with CAPS markers as listed in Table 1 followed by a restriction enzyme digest with BstXI . The wild type ATR contains a BstXI site, while atr-4 has lost this site. Digested amplicons were analyzed by gel electrophoresis, resolving wild type or mutant results in a 2% agarose gel.All GUS staining experiments were conducted as previously described , with microscopy performed using a Leica DMR differential interference contrast light microscope. Seedlings were grown on soaked gel media for 7 days, unless otherwise specified, collected, and subsequently fixed in 5 mL 90% acetone on ice for 20 to 30 minutes. Acetone was removed and seedlings were rinsed in 5 mL of rinse solution [50 mM NaPO4, 0.5 mM K3Fe6, and 0.5 mM K4Fe6]. Rinse solution was removed and seedlings were treated with 5 mL GUS stain [50 mM NaPO4, 0.5 mM K3Fe6, 0.5 mM K4Fe6, and 2 mM X-Gluc ], vacuum infiltrated for 5 minutes at room temperature, and incubated at 37°C for noted times. Stain solution was removed and seedlings were stored in 70% ethanol until analyzed using differential interference contrast microscopy. For CYCB1;1 GUS analyses, 60 total seedlings from each line and each treatment were scored for level of blue color at the root tip after 1 hour staining for GUS activity. To generate the SUV2:GUS line, the full length At1g45610 including the promoter was amplified from genomic DNA. PCR was performed using a high fidelity Taq polymerase, Phusion following the manufacturer’s instructions. This was assembled in the pGEMT EASY vector and then cloned into pBI101 containing GUS to create the SUV2:GUS fusion. The construct was electroporated into Agrobacterium tumefaciens strain Aglo, which was then used to transform Col-0 wt. F0 lines were screened on PNS with kanamycin for resistance. Kanamycin resistant lines were allowed to self and F1 lines were then screened again on kanamycin to identify homozygous lines. To generate the SUV2:GFP line, the full length At1g45610 including the promoter was amplified from genomic DNA. PCR was performed using a high fidelity Taq polymerase, Phusion following the manufacturer’s instructions. This was assembled in the pGEMT EASY vector and then cloned into pBI101 containing GFP to create the SUV2:GFP fusion. The construct was electroporated into Agrobacterium tumefaciens strain Aglo, which was then used to transform Col-0 wt. F0 lines were screened on PNS with kanamycin for resistance. Kanamycin resistant lines were allowed to self and F1 lines were then screened again on kanamycin to identify homozygous lines. Images were taken with a Lecia SP2 confocal microscope and nuclei were counter stained with Hoescht 33342. All pBI101 constructs were electroporated into the Algo strain of Agrobacterium tumefaciens using a Bio-Rad MicroPulser following the manufacturer’s instructions. Colonies were screened for transformants on LB medium containing Kanamycin and Streptomycin and colonies that appeared to contain the plasmid were grown and tested via PCR for the insert. Genomic DNA from the Agrobacterium line was grown overnight in 1mL LB medium containing Kanamycin and Streptomycin and at 28°C. Cells were pelleted and resuspened in 0.1 mL of ice-cold Solution I . Cells were incubated for 5 minutes at room temperature. To the cell suspension, 30 μL of phenol equilibrated with two volumes of solution II was added and vortexed gently for a few seconds. To this mixture, 150 μL of 3 M sodium acetate, pH 4.8 was added and the tube was shaken briefly. The tube was incubated at -20°C for 15 minutes, centrifuged for 3 minutes and then supernatant was poured into a new 1.5 mL tube. The tube was filled with ice-cold 95% ethanol, mixed by inversion and stored at – 80°C for 15 minutes. The tube was centrifuged for 3 minutes and the supernatant was discarded. To the pellet, 0.5 mL of 0.3 M sodium acetate, pH 7.0 was added and the pellet was resuspended. The tube was filled with ice-cold 95% ethanol and mixed well by inversion. The tube was then stored at -80°C for 15 minutes. The tube was centrifuged for 3 minutes, the supernatant was decanted and the tube was inverted on a paper towel to drain remaining supernatant. To the tube, 1 mL of icecold 70% ethanol was added, the tube was vortexed briefly and centrifuged for 1 minute. The pellet was resuspended in 50 μL of TE . DNA was then screened for by PCR using original cloning primers to detect complete SUV2:GUS or SUV2:GFP constructs. Flowering Col-0 wild type Arabidopsis thaliana was transformed with Agrobacterium lines that were confirmed to contain the appropriate plasmid. Agrobacterium lines carrying the appropriate plasmid were grown over night at 30°C in 500 mL of LB with appropriate antibiotics from a 5 mL starter culture. The overnight culture was pelleted at 5,000 rpm in a GSA rotor for 10 minutes.