LETTER TO THE EDITOR

The Nature of the Arabidopsis fon1 Mutations

Recently, we described three similar Arabidopsis mutants, floral organ number (fon)1-1, fon1-2, and fon1-3, that each exhibit an increased number of stamens and carpels (Huang and Ma 1997 ). Although these phenotypes resemble those conditioned by weak alleles of superman (sup), on the basis of results from the experiments outlined below, we originally concluded that SUP and FON1 are closely linked but distinct genes that share related functions (Huang and Ma 1997 ). However, in light of recently obtained data (Jacobsen and Meyerowitz 1997 ; S.E. Jacobsen and E.M. Meyerowitz, personal communication), we now find that a more plausible explanation of our results is that fon1 mutations are actually epigenetic alleles of SUP.

We determined that FON1 and SUP are closely linked during restriction fragment length polymorphism (RFLP) mapping experiments with 48 fon1 F₂ plants. These analyses indicated that FON1 is located on chromosome 3, ~15 cM from the marker GL1. Because previous studies had reported that GL1 is also closely linked to SUP, at a distance of 10.3 ± 3.2 cM (Schultz and Haughn 1991 ), we crossed fon1 and sup-1 mutants in an effort to determine whether or not the two mutations would complement each other. The phenotypes of the F₁ plants, which resembled those of fon1, suggested that fon1 mutations may in fact be alleles of SUP.

However, fon1 mutations are weakly semidominant, and mutations in interacting genes do not always complement each other (e.g., Atkinson 1985 ; Fuller 1986 ). Therefore, we further analyzed the F₂ progeny, finding that a little over 10% of the plants appeared to show no phenotype. DNA gel blot analyses indicated that about a quarter of these normal-appearing plants were SUP/SUP, which suggested that they carried a recombinant chromosome between FON1 and SUP. Because we assumed that a similar number of the F₂ progeny were FON1/FON1, we concluded that about half of the normal-appearing plants (just over 5% of the F₂ plants we analyzed) carried a recombinant chromosome. This would place FON1 a little over 5 cM from SUP, which is consistent with the RFLP results and the literature. The other half of the normal-looking F₂ plants (again, ~5% of the population) were inferred to be FON1/SUP transheterozygotes; we ascribed the absence of such normal plants in the F₁ population to its small size.

Further support for our original conclusion that FON1 and SUP are distinct genes came from experiments showing that the SUP sequences in fon1 mutants and wild-type plants (Sakai et al. 1995 ) are the same (Huang and Ma 1997 ).

Recently, however, Jacobsen and Meyerowitz 1997 reported their analyses of several clark kent (clk) mutants, which have phenotypes very similar to those of fon1 plants. They showed that the clk mutants also map very close to SUP and demonstrated that normal-looking revertants appear at a high frequency. Furthermore, although the clk mutants do not have nucleotide changes in the SUP gene, increased levels of methylation at the SUP locus correlate with reductions in SUP gene expression. On the basis of their data, they concluded that the clk mutants carry epigenetic alleles of SUP (Jacobsen and Meyerowitz 1997 ).

Because the fon1 and clk mutants are similar, we provided seeds carrying the fon1-2 and fon1-3 alleles to Drs. Jacobsen and Meyerowitz. Their analyses show that fon1-2 and fon1-3 have SUP methylation patterns similar to those found in clk mutants (Jacobsen and Meyerowitz 1997 ). More recently, they have found that a cross between fon1 and clk mutants produces progeny with a fon1/clk phenotype (S.E. Jacobsen and E.M. Meyerowitz, personal communication).

In light of these new findings, we would like to state that the phenotypes and SUP sequence information now suggest that the fon1 mutations are also epigenetic alleles of SUP. This conclusion is reinforced by the phenotypes of double mutants of fon1 with other floral mutations, which are similar to, albeit weaker than, those of the available corresponding double mutants with sup. The normal plants that appear among the F₂ progeny of the cross between fon1 and sup mutants (Huang and Ma 1997 ) are probably the result of reversion of the epigenetic alleles.

The isolation of many epigenetic alleles at the SUP locus is quite unusual, although this could be because similar epigenetic alleles at other loci have not been recognized as such. Recent studies of mutants and/or transgenic plants have revealed that reduced DNA methylation is associated with developmental abnormalities, including floral defects similar to those observed in sup and agamous mutants (Finnegan et al. 1996 ; Kakutani et al. 1996 ; Ronemus et al. 1996 ). Because of the close association between DNA methylation and epigenetic alleles, these defects could be due to epigenetic alleles at specific developmental loci, rather than a direct consequence of DNA hypomethylation. It is also interesting to note that sup epigenetic alleles are associated with DNA hypermethylation and not with hypomethylation (Jacobsen and Meyerowitz 1997 ), indicating that there might be more than one way to generate epigenetic alleles in plants.

ACKNOWLEDGMENTS

We would like to express our appreciation to Drs. Jacobsen and Meyerowitz for sharing unpublished results.

REFERENCES

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Fuller, M.T. (1986). Genetic analysis of spermatogenesis in Drosophila: The role of testis-specific ß-tubulin and interacting genes in cellular morphogenesis. In Gametogenesis and the Early Embryo, I.G. Gall, ed (New York: Liss), pp. 19-41.

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