Epigenetic regulation, somatic homologous recombination, and abscisic acid signaling are influenced by DNA polymerase epsilon mutation in Arabidopsis

Abstract

Based on abscisic acid (ABA) inhibition of seed germination and seedling growth assays, we isolated an ABA overly sensitive mutant (abo4-1) caused by a mutation in the Arabidopsis thaliana POL2a/TILTED1(TIL1) gene encoding a catalytic subunit of DNA polymerase epsilon. The dominant, ABA-insensitive abi1-1 or abi2-1 mutations suppressed the ABA hypersensitivity of the abo4-1 mutant. The abo4/til1 mutation reactivated the expression of the silenced Athila retrotransposon transcriptional silent information (TSI) and the silenced 35S-NPTII in the ros1 mutant and increased the frequency of somatic homologous recombination (HR) approximately 60-fold. ABA upregulated the expression of TSI and increased HR in both the wild type and abo4-1. MEIOTIC RECOMBINATION11 and GAMMA RESPONSE1, both of which are required for HR and double-strand DNA break repair, are expressed at higher levels in abo4-1 and are enhanced by ABA, while KU70 was suppressed by ABA. abo4-1 mutant plants are sensitive to UV-B and methyl methanesulfonate and show constitutive expression of the G2/M-specific cyclin CycB1;1 in meristems. The abo4-1 plants were early flowering with lower expression of FLOWER LOCUS C and higher expression of FLOWER LOCUS T and changed histone modifications in the two loci. Our results suggest that ABO4/POL2a/TIL1 is involved in maintaining epigenetic states, HR, and ABA signaling in Arabidopsis.

Figure 1.

ABA Sensitivity of the abo4-1 Mutant. (A) ABA sensitivity of abo4-1 seedlings grown on MS media containing different concentrations of ABA. Five-day-old seedlings grown on MS medium were transferred to MS medium containing different concentrations of ABA, respectively, and cultured for another 7 d before taking pictures. (B) The seed germination sensitivity of abo4-1 to ABA. Wild-type and abo4-1 seeds were directly sowed on MS medium or MS medium containing 0.3 μM ABA, respectively. After being kept at 4°C for 3 d, the plates were removed to a growth chamber and cultured for 7 d before taking pictures. (C) The statistic data of seed germination of wild-type and abo4-1 on MS medium containing different concentrations of ABA. The experimental conditions were the same as in (B). Values are means ± se, n = 3 independent experiments. At each time, >100 seeds were counted. (D) The dominant mutations of the negative regulators ABI1 and ABI2 block the ABA sensitivity of abo4-1. The seeds of wild-type, abo4-1, abi1-1, abi2-1, abo4-1 abi1-1, and abo4-1 abi2-1 double mutants were germinated on MS medium containing no ABA or 1 μM ABA. The pictures were taken after seedlings were grown for 7 d in a growth chamber. (E) Mapping-based cloning of ABO4. The ABO4 locus was mapped between two BAC clones F24B9 (recombinants, 7 of 1802) and T27G7 (recombinants, 3 of 1802). Further mapping delimited the ABO4 locus to a region within BAC T23G18. A point mutation (G4171A, in the 13th exon) was found in AT1G08260/POL2a/TIL1. A T-DNA insertion (abo4-2, SALK_096341) line was obtained with a T-DNA inserted at position 3972 (in the 12th exon). (F) Comparison of ABA sensitivity of wild-type and abo4-2 seedlings. The seedlings were first grown on MS medium for 5 d. abo4-2 seedlings were picked up according to its grown phenotype and were, together with the wild type, transferred to MS medium containing different concentrations of ABA. Here, we only show the seedlings grown on MS medium containing 60 μM ABA for 7 d. The similar growth phenotypes were observed when abo4-2 seedlings were grown on other concentrations of ABA (10, 20, 30, and 50 μM, respectively).

Figure 2.

abo4 Mutants Are Sensitive to DNA Damage. (A) Comparison of the wild type and abo4-1 seed germination on MS medium containing no (left) or 125 ppm (right) MMS. Seedlings were grown for 10 d on MS medium containing no MMS or 125 ppm MMS. (B) Seedling growth sensitivity of wild-type gl1, abo4-1 (gl1), the wild type, and abo4-2 in MS liquid medium supplemented with different concentrations of MMS. Five-day-old seedlings were removed to the MS liquid medium containing different concentrations of MMS and cultured for 10 more days before taking pictures. (C) Relative fresh weight of wild-type, abo4-1, and abo4-2 seedlings grown on 75 and 125 ppm MMS. The fresh weight of wild-type, abo4-1, and abo4-2 seedlings grown on MS agar medium for 10 d was used to compare with the fresh weight of seedlings grown on 75 and 125 ppm MMS, respectively. For each data point, 30 seedlings were collected and weighed. Data are the mean ± sd of three independent experiments. (D) The segregation of F1 seedlings from heterozygous abo4-2 mutant lines crossed with abo4-1 grown on MS medium containing 100 ppm MMS (MMS sensitive to tolerant seedlings: 58/58). We randomly picked up 20 sensitive seedlings and checked by PCR, and all were heterozygous plants of abo4-1 and abo4-2. Arrows point to the segregated mutants. (E) abi1 and abi2 mutations did not improve the DNA damage sensitivity caused by MMS in abo4-1. The similar experiments were done as in (B) for abo4-1 abi1-1 and abo4-1 abi2-1 double mutant seedlings. (F) Relative fresh weight of wild-type, abi1-1, abi2-1, abo4-1 abi1-1, and abo4-1 abi2-1 seedlings grown on 75 and 125 ppm MMS. The fresh weight of wild type, abi1-1, abi2-1, abo4-1 abi1-1, and abo4-1 abi2-1 seedlings grown on MS agar medium for 10 d was used to compare with the fresh weight of seedlings grown on 75 and 125 ppm MMS, respectively. For each data point, 30 seedlings were collected and weighed. Data are the mean ± sd of three independent experiments. (G) abo4-1 seedlings were more sensitive to UV-B light than the wild type. Five-day-old seedlings of the wild type and abo4-1 in the same plate were treated with different amounts of UV-B light and then grown in light (for 1 and 5 kJ/m²) or first in dark for 2 d and then in light (for 3 kJ/m²). The pictures were taken after seedlings were grown in light for another 5 d. (H) Comparison of the gene expression in the wild type and abo4-1 under normal and MMS (100 ppm) treatment using real-time qRT-PCR. GR1, RAD51, BRCA1, MRE11, KU70, MSH2, and MSH6 were selected for comparison. The transcripts of the wild type in normal condition were used as a standard for normalization. (I) Comparison of the transcripts of GR1, RAD51, and BRCA1 by qRT-PCR between the wild type and abo4-1 under normal and UV-B (3 kJ/m², in dark for 6 h) treatment. In both (H) and (I), three independent biological replications were performed for each experiment. The level of 18S rRNA was used as an internal control. Error bars indicate ±sd.

Figure 3.

The abo4 Mutation Increases Cell Cycle–Related Gene Expression. (A) The representative examples of GUS staining. CYCB1;1 promoter-GUS was expressed higher in shoot and root meristems of abo4-1 and abo4-2 than the wild type, and its expression was highly induced by MMS treatment but not affected by ABA. (B) The relative expression levels of CYCB1;1, CYCA2;1, CDC2A, and H4b in the wild type and abo4-1 analyzed by qRT-PCR. The expression of each gene in the wild type was used as a standard. The representative results were from one of three independent experiments. Error bars indicate ±sd.

Figure 4.

The Effects of abo4 Mutation and ABA on HR. (A) The diagram of GUS reporter constructs in direct (1415, left) and inverted repeat (1406, right) used in this study (adapted from Lucht et al., 2002). (B) Typical patterns of GUS staining in abo4-1, abo4-2, and the wild type carrying GUS reporter lines 1406 and 1415, respectively, under normal growth conditions. (C) and (E) Quantitative analysis of HR in ∼100 plants of abo4-1 and the wild type carrying reporter line 1415 (C) and 1406 (E), respectively, under normal and ABA treatment conditions. The number of the GUS staining sectors was counted for each plant. The number of plants assayed was 115 for each assay. The proportions of plants showed frequency distribution of a given number of blue GUS spots in 1415 and 1406 populations. (D) and (F) The average GUS spot number per seedling in ∼100 abo4-1 and wild-type seedlings under ABA and normal conditions. The number of plants assayed was 105 for each assay. Two independent experiments were performed. Error bars indicate ±sd. The abo4-1 showed a 48-fold increase in the number of blue sectors over the reporter line 1415 and 61-fold increase over the reporter line 1406 than the wild type under normal conditions.

Figure 5.

ABA Treatment Increases DNA Breaks and Upregulates GR1 and MRE11 Expression. (A) The TUNEL assay under normal or ABA-treated conditions. Under the normal growth condition, TUNEL staining was negative in both the wild type and abo4-1 mutant (−ABA). Under ABA (30 μM for 3 d) treatment, only abo4-1 showed many TUNEL (bright green fluorescent signal) nuclei, but the wild type still showed negative TUNEL nuclei. The 4',6-diamidino-2-phenylindole (DAPI) staining (blue color) was used to indicate the nuclei. (B) Comparison of GR1, MRE11, KU70, RAD51, BRCA1, and NSH2 expression by qRT-PCR under ABA treatment. The expression of GR1 and MRE11 was increased, and KU70, RAD51, and BRCA1 decreased by ABA treatment (30 μM) especially in the later time points in both abo4-1 and the wild type. The expression of MSH2 was not influenced by ABA. The transcripts of each gene under normal condition were used a comparison standard. The representative data were from one of three independent experiments showing similar results. Error bars indicate ±sd.

Figure 6.

Releasing of TGS of 35S-NPTII in ros1 and TSI by abo4 Mutation Independent on DNA Methylation. (A) abo4 mutation reactivated the silenced 35S-NPTII but did not affect the silenced RD29A-LUC in ros1-1 mutant. The seeds of wild-type_RD29A-LUC, ros1-1_RD29A-LUC, and abo4-1 ros1-1_RD29A-LUC carrying RD29A-LUC transgene were germinated and grown on MS medium (left plate) or MS medium supplemented with 50 mg/L kanamycin (middle plate) for 2 weeks. Both wild-type_RD29A-LUC and abo4-1 ros1-1_RD29A-LUC seedlings showed the kanamycin-resistant phenotype from expression of the NPTII gene, while ros1-1_RD29A-LUC seedlings were sensitive to kanamycin due to transgene silencing. Two-week-old seedlings were treated with 100 μM ABA for 5 h to induce the expression of RD29A-LUC. LUC expression was only detected in wild-type_RD29A-LUC but not in ros1-1_RD29A-LUC and ros1-1 abo4-1_RD29A-LUC double mutants (right plate), indicating that abo4 mutation did not reactivate the silenced RD29A-LUC in ros1-1. (B) RNA gel blot analysis of endogenous RD29A and 35S-NPTII expression under ABA treatment. Total RNAs extracted from different seedlings treated or not treated by 100 μM ABA for 5 h were used for hybridization. A stress-inducible marker gene, COR47, was used as positive control to indicate that ABA treatment equally induced its expression in wild-type_RD29A-LUC, ros1-1_RD29A-LUC, and ros1-1 abo4-1_RD29A-LUC. RD29A expression was only detected in the wild type by ABA treatment, while NPTII transcripts were detected in both wild-type_RD29A-LUC and ros1-1 abo4-1_RD29A-LUC. The endogenous RD29A and the transgene copy of 35S-NPTII were silenced in ros1-1_RD29A-LUC. TUBULIN was used as a loading control. (C) abo4 mutation did not release the TGS of RD29A-LUC. RNA gel blot analysis of RD29A-LUC expression in wild-type_RD29A-LUC, ros1-1_RD29A-LUC, and ros1-1 abo4-1_RD29A-LUC under 300 mM NaCl for 5 h. LUC transcripts were only detected in the wild type and not in ros1-1 and ros1-1 abo4-1. (D) TSI expression was enhanced in the abo4-1 and abo4-2 mutants. RNA gel blot analysis of TSI expression in ddm1 was used as a control for comparison. (E) abo4 mutation did not affect the DNA hypermethylation in the transgene and endogenous RD29A promoter in ros1-1_RD29A-LUC. Genomic DNAs extracted from wild-type_RD29A-LUC, ros1-1_RD29A-LUC, and ros1-1 abo4-1_RD29A-LUC were digested with DNA methylation-sensitive enzymes MluI (A^mCG^mCGT) and BstUI (^mCG^mCG) as described (Gong et al., 2002) and hybridized with ³²P-labeled LUC (for transgene RD29A promoter) or RD29A (for endogenous RD29A promoter). (F) Methylation status of centromeric DNA (Cen180) or TSIs was neither influenced by abo4 mutations nor by ABA treatment. Genomic DNAs were extracted from (1) the wild type; (2) abo4-1; (3) abo4-2; ABA−, without ABA treatment; ABA+, seedlings were treated with 10 μM ABA for 7 d and digested by DNA methylation-sensitive restrictive enzymes HpaII (^mC^mCGG methylation), MspI (^mCCGG methylation), and Nla III (^mCATG methylation). The membranes were hybridized with ³²P-labeled Cen 180 bp or TSI fragment, respectively. (G) Both ABA and MMS treatment increased the TSI transcripts more in abo4-1 than the wild type. Seven-day-old seedlings were treated with MMS (M1, 50 ppm; M2, 100 ppm) for 7 d or treated with ABA (A1, 5 μM; A2, 10 μM) for 7 d. RNA gel blot analysis was performed using TSI as probe. RNAs were used as loading control. (H) qRT-PCR analysis of TSI expression in the wild type and abo4-1 treated by 5 μM ABA or 50 ppm MMS for 7 d. Untreated wild type was used as a standard control. Three biological replications were performed for each experiment. Error bars indicate ±sd.

Figure 7.

Growth phenotypes of abo4 mutants. (A) Seedling comparison of wild-type, abo4-1, abo4-2, and abo4-1 abo4-2 heterozygous plants on MS agar plates. Please notice that abo4-2 shows a smaller phenotype, and abo4-1 abo4-2 heterozygous plants show abo4-1 phenotype. (B) Comparison of wild-type, abo4-1, abo4-2, and abo4-1 abo4-2 heterozygous plants grown in soil. (C) The F1 seedlings of heterozygous abo4-2 crossed with abo4-1. The ratio of abo4-1 to wild-type phenotype is ∼1:1 (54:56). abo4-2/abo4-1 mutants showing abo4-1 phenotypes are indicated with arrows. (D) Comparison of the rosette leaves of wild-type (gl1), abo4-1(gl1), wild-type (Col), and abo4-2 (Col) before flowering. abo4-1 is in Col gl1 background, and abo4-2 is in Col background. Because there is no growth difference in Col gl1 and Col except for Col gl1 with no trichomes, we only used Col gl1 as control in later analysis. (E) Comparison of siliques in wild-type, abo4-1, and abo4-2. Please notice that no seeds were set in abo4-2 siliques, and fewer seeds were set in abo4-1 than the wild type. (F) to (H) Comparison of stems of the wild type (F), abo4-1 (G), and abo4-2 (H). Here, we only show abo4-1 and abo4-2 stems with fasciation phenotype. However, most of the abo4-1 and abo4-2 stems grew normally. (I) and (J) Comparison of flowers of the wild type (I) and abo4-1 (J). (K) and (L) Comparison of roots of the wild type (K) and abo4-1 (L). The root elongation zone is shorter in abo4-1 than the wild type. Bars = 5 mm. (M) and (N) Comparison of SAMs of the wild type (M) and abo4-1 (N). Three classical zones were clearly observed in the SAM of the wild type but not in the SAM of abo4-1. Bar = 100 μm. (O) qRT-PCR analysis of FLC, FT, and AP1 expression. In each case, RNAs from the wild type were used as a standard control for normalization for each RNA. The representative data were from one of three independent experiments showing similar results. The level of 18S rRNA was used as an internal control. Error bars indicate ±sd. (P) ChIP analysis of histone H3K27me3 and H3K4me2 modifications in the first intron of FLC (the same primers are used as in Cao et al., 2008) and in the FT promoter region by qRT-PCR. Three independent immunoprecipitations were performed for each experiment. The immunoprecipitated DNA was quantified by real-time qRT-PCR. We used ACTIN as internal controls for the H3K4me2 level and TA3 for the H3K27me3 level. Error bars indicate ±sd.