Fig 1.
MMS19 interacts with the Fe-S assembly complex components CIA1 and CIA2 and the catalytic subunits of DNA polymerases in the cytoplasm.
(A) MMS19 interacts with CIA1 and CIA2 in vivo as determined by co-IP. (B) MMS19 interacts with ICU2 in vivo. (C) Gel filtration analysis of MMS19, CIA1, and CIA2. The protein extracts were isolated from flowers of transgenic plants (MMS19-Myc, CIA1-Flag, and CIA2-Flag) in the wild-type or mms19 mutant background and were loaded onto a Superose 6 10/300 GL column. The eluted fractions were run on an SDS-PAGE gel and subjected to Western blotting. The fraction numbers and sizes of standard proteins are shown. The subcellular localization of MMS19 (D), CIA1 (E), CIA2 (F), NAR1 (G), and ICU2 (H) as determined by nuclear-cytoplasmic fractionation. T: total extraction proteins, C: cytoplasmic proteins, N: nuclear proteins. MMS19, CIA1, and CIA2 mainly localized in the cytoplasm while NAR1 and ICU2 localized both in the cytoplasm and the nucleus. Histone H3 and UGPase were used as a nuclear marker and a cytoplasmic marker, respectively.
Table 1.
Mass spectrometric analyses of MMS19, CIA1, and CIA2 affinity.
Fig 2.
Silencing of 35S-NPTII transgene and endogenous transposable elements and other loci is affected in the mms19 mutant.
(A) The effect of mms19 on the silencing of RD29A-LUC and 35S-NPTII transgenes. Each genotype harboring the RD29A-LUC and 35S-NPTII transgenes was grown on MS medium for 14 days followed by cold treatment for 2 days at 4 °C. The treated seedlings were sprayed with luciferin for luminescence imaging. The seedlings were grown on MS medium supplemented with 150 mg/L kanamycin for 20 days and photographed. (B) The mms19 and abo4 mutants release the silencing of transposable elements. The transcript levels of the transposable element genes TSI, AT2G11780, and AT3G32195 were detected in the wild type, mms19-2 and its complementation line, and abo4 by quantitative RT-PCR. ACT7 was used as an internal control for normalization. Quantitative RT-PCR experiments were biologically repeated three times with similar results. Showing is the result of three technical replicates from one representative experiment.
Fig 3.
The effect of mms19 on the transcriptome as determined by RNA-seq analyses.
(A) Differentially expressed genes in mms19 and abo4 mutants relative to the wild type are shown by Venn diagrams. (B) Differentially expressed genes in mms19 and abo4 mutants relative to the wild type are shown by heat maps. (C) Differentially expressed TEs in mms19 and abo4 mutants relative to the wild type are shown by Venn diagrams. (D) Gene Ontology (GO) analysis of co-upregulated genes in mms19 and abo4 mutants. The lengths of bars represent statistical values of gene enrichment in the indicated biological processes. The biological processes are listed only when their genes are significantly (P<0.05) enriched.
Fig 4.
The mms19 mutants are sensitive to DNA damage and constitutively express a high level of DNA repair-related genes.
(A) The mms19 mutants are more sensitive to UV-B treatment than the wild type. Five-day-old seedlings of the wild type and mms19 mutants were treated with UV-B light (1 kJ/m2) for different times and then grown with 16-h-light and 8-h-dark at 22° for 5–7 days before being photographed. (B) The sensitivity of the mms19-2 mutant to UV-B treatment was rescued by the MMS19-Myc transgene in T2 transgenic plants. (C) The mms19 mutants are more sensitive than the wild type to the DNA-damaging reagent methyl methanesulfonate (MMS). Quantitative RT-PCR results indicate that DNA repair-related genes were up-regulated in ros1icu2 (D), abo4 (E), and mms19 (F) mutants compared to their respective control plants. The seedlings were grown on MS medium plates for ~10 days under long-day-condition and then harvested for RNA isolation. Quantitative RT-PCR experiments were biologically repeated with similar results, and three technical replicates of one representative experiment were shown. Error bars represent the standard deviation (SD).
Fig 5.
The mms19 mutants cause an early flowering phenotype by affecting the expression of flowering-related genes.
(A) The early flowering phenotype of mms19 mutants in standard long-day conditions (16-h-light and 8-h-dark at 22°C). (B) The early-flowering phenotype was restored by the MMS19-Myc construction in mms19-2. (C) The statistics of total leaf numbers upon flowering under long-day conditions in the wild type, the mms19 mutants and the complementation lines. T2-5 and T2-6 were two randomly selected individual MMS19-MYC transgenic lines in T2 generation. At least 30 individual plants were counted. Error bars stand for SD. Asterisks indicate significant difference as determined by the t-test (P<0.05). Numbers of rosette and cauline leaves are indicated by blue and red bars, respectively. (D) Leaf numbers under long-day conditions with or without vernalization. (E) Leaf numbers under short-day conditions. (F), (G) and (H) The effect of mms19, abo4, and icu2 on the expression of flowering-related genes as determined by quantitative RT-PCR. ACT7 was amplified as an internal control. The quantitative RT-PCR experiments were biologically repeated for three times and indicated similar results. A representative repetition is shown. Error bars represent SD. Asterisks show significant difference as determined by the t-test (P<0.05).
Fig 6.
The mms19 mutant incorporates a reduced level of Fe in ICU2 and is sensitive to Fe absence.
(A) Fe content of ICU2 in the wild type and mms19. The protein extraction was affinity purified by anti-Myc antibody-coupled beads and eluted three times by 0.1 M ammonium hydroxide. The three elutions were combined and subjected to Fe content detection by ICP-MS (Themo ICP-MS XII). The affinity purified ICU2-Myc in the wild type and the mms19 mutant was separated by SDS-PAGE and subjected to silver staining and western blotting as controls. The results of three biological repeats are indicated. Error bars show the SD. Asterisks indicate significant differences as determined by the t-test (P<0.05). (B) 55Fe incorporation into ICU2 in the wild type and the mms19 mutant. The same affinity purification method was used as in (A), and the radioactivity of 55Fe in ICU2 was measured by liquid scintillation counting. Triplicates were performed. Error bars show the SD. Asterisks indicate significant differences as determined by the t-test. (C) The activities of aldehyde oxidase (AO) isozymes in wild-type and mms19 mutant seedlings. The purple bands indicated by arrows represent the activities of AO proteins. The staining by Ponceau S is shown as an equal protein loading control. (D) The mms19 and abo4 seedlings were more sensitive than wild-type seedlings to reduced FeSO4 concentrations. Seedlings were photographed after they had grown for 10 days on MS medium plates with different FeSO4 concentrations. 100%, 50%, 25%, and 12.5% represent 2.78, 1.39, 0.70, and 0.35 g/l of FeSO4.7H2O in MS medium.