MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required in stem cell regulation and to maintain developmentally regulated gene silencing

Abstract

Maintenance of stem cells in the Arabidopsis thaliana shoot meristem is regulated by signals from the underlying cells of the organizing center, provided through the transcription factor WUSCHEL (WUS). Here, we report the isolation of several independent mutants of MGOUN1 (MGO1) as genetic suppressors of ectopic WUS activity and enhancers of stem cell defects in hypomorphic wus alleles. mgo1 mutants have previously been reported to result in a delayed progression of meristem cells into differentiating organ primordia (Laufs et al., 1998). Genetic analyses indicate that MGO1 functions together with WUS in stem cell maintenance at all stages of shoot and floral meristems. Synergistic interactions of mgo1 with several chromatin mutants suggest that MGO1 affects gene expression together with chromatin remodeling pathways. In addition, the expression states of developmentally regulated genes are randomly switched in mgo1 in a mitotically inheritable way, indicating that MGO1 stabilizes epigenetic states against stochastically occurring changes. Positional cloning revealed that MGO1 encodes a putative type IB topoisomerase, which in animals and yeast has been shown to be required for regulation of DNA coiling during transcription and replication. The specific developmental defects in mgo1 mutants link topoisomerase IB function in Arabidopsis to stable propagation of developmentally regulated gene expression.

Figure 1.

s3801 Suppresses 35S:WUS-GR Induced Inhibition of Differentiation. (A) to (C) Induction of WUS-GR by dexamethasone (dex) results in arrest of differentiation in the wild type background ([B], compare with mock treatment in [A]). This developmental arrest is suppressed by the s3801 mutation (C). (D) and (E) Eight-day-old s3801 seedlings (E) display retarded and pointed leaves in comparison to the Ler wild type (D). (F) and (G) The vegetative s3801 shoot meristem (G) is disorganized in comparison to the wild type (F). Bars = 0.5 mm in (D) and (E) and 200μm in (F) and (G).

Figure 2.

mgo1 Meristem Phenotypes. (A) and (B) Inflorescences of wild-type (A) and mgo1-4 plants (B). The mutant displays fasciation and fragmentation of the apex. Phyllotaxis and internode spacing is variable in mgo1-4 (B) in contrast with the stereotypic arrangement in the wild type (A). (C) and (D) Inflorescence shoot meristems of wild-type (C) and mgo1-4 plants (D). The mgo1-4 inflorescence meristem displays multiple independent meristems (asterisks), each of which is generating floral primordia at its flanks. (E) to (H) WUS:GUS and CLV3:GUS expression. In mgo1-4 mutants ([F] and [H]), expression of both stem cell niche markers is fragmentized into multiple domains arranged in a line, in contrast with the wild type ([E] and [G]). (I) and (J) Confocal image of root meristems of 5-d-old seedlings of Columbia (Col) wild type (I) and mgo1-7 (J) plants. Dead cells, which accumulate propidium iodide, are marked by an arrow. (K) and (L) The number of cells expressing the CYCB1;1:GUS reporter is strongly reduced in 5-d-old primary roots of mgo1-4 (L) versus Ler (K). Bars = 5 mm in (A) and (B), 200 μm in (C) and (D), 50 μm in (E) to (H), 200 μm in (I) and (J), and 100 μm in (K) and (L).

Figure 3.

Genetic Interactions of MGO1 and WUS. (A) to (E) Eleven-day-old seedlings. In wild-type (A), mgo 1-4 (B), and wus-7 (D) seedlings, the first leaves have been formed. In the severe wus-1 mutant (C), intermediate wus-6 mutant (inset), and in the mgo 1-4 wus-7 double mutant (E), the primary shoot meristem has terminated without leaf formation. (F) and (G) Comparison of wus-1 (F) and wus-6 (G) 45-d-old plants. Adventitious shoots in wus-1 terminated prematurely in an aerial rosette, whereas wus-6 produces an indeterminate shoot with many flowers. (H) wus-6 mgo1-4 double mutants rarely formed adventitious shoots and never produced flowers. (I) Comparison of wus-1, wus-7, and mgo 1-4 wus-7 40-d-old plants. mgo1-4 wus-7 double mutants formed determinate adventitious shoots with a few defective flowers similar to wus-1, whereas wus-7 single mutants produced indeterminate shoot with many flowers. (J) to (M) Flower phenotype of wus-1 (J), wus-6 (K), wus-7 (L), and mgo1-4 wus-7 (M) plants. (N) Quantitative RT-PCR experiment showing mRNA expression levels of WUS. Asterisk represents significant difference of WUS mRNA compared with the wild type (P < 0.05). (O) No changes in MGO1 transcript levels are detectable by RT-PCR after induction of 35S:WUS-GR plants with dexamethasone (D) compared with mock treated plants (M). ACT7 (actin7) transcript was used as control. Bars = 2.5 mm in (A) to (E), 2 cm in (F) to (H), 1 cm in (I), and 2 mm in (J) to (M).

Figure 4.

The wus-7 fas1-1 Double Mutant. (A) to (E) Phenotype of 10-d-old seedlings. In wild-type (A) and wus-7 (B) seedlings, the first leaves have been formed. In wus-1 (C), the primary shoot meristem has terminated without leaf formation. In fas1-1 (D) seedlings, leaf formation is strongly delayed and leaves are malformed. fas1-1 wus-7 (E) seedling displaying an empty apex (arrowhead) instead of a shoot meristem. (F) to (I) Comparison of 45-d-old plants. Adventitious shoots in wus-1 (G) terminated prematurely in an aerial rosette, whereas wus-7 (F) and fas1 (I) shoots are indeterminate. fas1-1 wus-7 (H) plants terminated prematurely like wus-1. Bars = 2.5 mm in (A) to (E) and 2 cm in (F) to (I).

Figure 5.

Combinations of mgo1-4 with Chromatin Factor Mutants. (A), (B), (E), (F), (I), and (J) Phenotype of 16-d-old seedlings of single and double mutants with the genotypes indicated. (C), (D), (G), (H), (K), and (L) About 5-week-old single and double mutants. (E) to (H) pkl-15 mgo1-4 double seedlings are dwarfed plants (F) with sessile, foliage leaves, which later develop enlarged and misshapen shoot apices (arrow in [G]). (I) to (K) syd-2 mgo1-4 double mutant seedlings do not show primary leaves at 16 d (J), unlike each single mutant ([B] and [I]). At later stages, double mutants produce narrow, serrated leaves and enlarged shoot meristems (K). (L) syd-2 adult 40-d-old plant with curled leaf (arrow, inset). (M) and (N) Nineteen-day-old mgo1-7 lhp1-3 seedlings (N), in comparison with the lhp1-3 single mutant (M). (O) and (P) Phenotype of 19-d-old mgo1-4 clf-2 double mutants (P): leaf curling is more severe than in either single mutant ([B] and [O]). Bars = 2 mm in (A) to (C), (E), (F), (I), (J), and (Q) to (T), 2 cm in (D), (H), and (L), 0.5 mm in (G), and 1 mm in (K).

Figure 6.

Deregulated Gene Expression in mgo1 (A) to (D) An early arising mgo1-4 flower is shown in (B), a later arising flower with stigmatic tissue and ectopic ovules on outer whorl organs in (C), and a terminal mgo1-4 flower in (D). (E) and (F) Ectopic carpelloid tissue formation (cf. to [C] and [D]) is repressed in ag-1 mgo1-4 (F) plants. (G) to (M) Expression of pAG:I-GUS (blue) is ectopically activated in random sectors (arrowheads) in mgo1-4 in contrast to the wild type in rosette leaves ([G] and [H]), inflorescences ([I] and [J]), and flowers ([K] and [L]). A revertant sector (arrowhead) is shown in (M). (N) and (O) Expression of pBP:GUS (blue) is detected in cotyledonary vasculature and at the tips of the rosette leaves of mgo1-4 (N) but not in the wild type (O). Bars = 2 mm in (A) and (B), 1 mm in (C) and (D), 2 mm in (E) to (J), and 1 mm in (K) to (O).

Figure 7.

MGO1 Gene Structure and Expression. (A) Gene structure of MGO1/TOP1α and identified mutations. (B) to (E) Expression of MGO1 examined by in situ hybridization on wild-type inflorescence tissues. MGO1 transcript (brown-reddish color) preferentially accumulates in the inflorescence meristem (im) and in inner whorls of floral buds (B), and in a stage 10 flower (C) in petals (pe), stamens (st), and inner margin of the gynoecium (gy). Sense controls are in (D) and (E). (F) and (G) MGO1 transcript accumulates throughout mid-stage embryos (F). Sense control is in (G). Bar = 100 μm.