NATURE|Vol 437|1 September 2005 BRIEF COMMUNICATIONS ARISING

E1

Lolle et al. suggest that non-mendelian inheri-tance in Arabidopsis thaliana might be attrib-utable to an ancestral RNA-sequence cache1,whereby the RNA genome of previous genera-tions causes a high rate of reversion of theplant’s mutant hothead (hth) and erecta (er)genes. Here I describe a ‘distributed genome’model that also explains their results, in whichmutant hth DNA is restored by homologoussequences present in the genome itself. Thismodel has implications for the generation ofdiversity without mating.

DNA-homology searches of the Arabidopsisgenome based on the 21 nucleotides surround-ing hth-4, hth-8, hth-10 and er reveal the pres-ence of short, perfectly homologous DNAstretches (known as ‘reverting sequences’) thatinclude nucleotides needed to correct thesemutations (Fig. 1). There are also many exam-ples of short homology in genes tested for poly-morphism (including GL1, UFO and GAPC).

These sequences might be transcribed intoshort RNA molecules directed against otherchromosomal loci by the cellular machinery,perhaps with the involvement of DRD1(ref. 2), producing short RNA–DNA hybridswith potential mismatches that can be cor-rected by mismatch repair3. Consistent withthis model, the hth-4 allele — with 6 revertingsequences of 13–15 nucleotides each — has alower reversion frequency than hth-10, whichhas 24 reverting sequences of 13–18nucleotides. As a result of such differences innumber, as well as differences in the produc-tion of short RNA, some sequences might bechanged more than others.

These short sequences should also produceforward mutations, so it is important to mea-sure forward-mutation frequency for severalloci. Also, of the several short sequences avail-able for reversion, some might express moreshort RNA in the male gamete, explaining thepreferential transmission of reverted allelesthrough pollen. The messenger RNA of the cor-responding gene may competitively hybridizewith a small length of RNA and prevent itsinteraction with DNA but, owing to the short-ness of the base-paired sequence, the mRNAwould not be totally inactivated and so wouldnot produce a mutant phenotype. As both non-sense and missense alleles can cause a reductionin transcription compared with the wild-typeallele4, there would be more opportunity forreversion to wild type than for forward muta-tion, which would account for the shielding ofthe genome against forward mutations.

Conversion to neutral alleles could alsooccur by this mechanism, but again somesequence stretches might be more effectivethan others. Lolle et al. did not find any neutral

mutation in nine reverted HTH genes1. Even ifthe activity of all sequence stretches were com-parable, the active reversion at any site shouldbe independent of events at other sites: there-fore, the frequency of a neutral mutationamong revertants is expected to be around 1%,lower than the level of detection in Lolle et al.1.

The proposed sequence-mediated reversion

frequency could be boosted by another mech-anism not described by Lolle et al.1. The hthmutant cuticles have increased cellular perme-ability compared with the wild type5. I suggestthat the hth embryo sac is also more porousthan the HTH embryo sac, causing DNA in theHTH/hth heterozygote to enter the hth embryosac from the two degraded HTH spores andbecome ‘archived’, as in the P22 phage6,7.Although the increased cellular permeabilitywould allow the hth gametophyte to obtainHTH molecules in the F1 generation, in subse-quent generations these DNAs need not repli-cate; the endogenous reverting sequence mightprovide a basal level of reversion.

Lolle et al. propose that metabolic stress inhth increases its reversion frequency1, whichmight increase information transfer betweenselected short sequences to alter DNA and create genetic diversity.Abed ChaudhuryCSIRO Plant Industry, GPO Box 1600, ACT 2601,Australiae-mail: abdul.chaudhury@csiro.au

1. Lolle, S. J., Victor, J. L., Young, J. M. & Pruitt, R. E. Nature434, 505–509 (2005).

2. Matzke, M. A. & Birchler, J. A. Nature 455, 24–34 (2005).3. Watson, J. D., Losick, R., Bell, S. P., Gann, A. & Levine, M.

Molecular Biology of the Gene (Cummings, San Francisco,2003).

4. Mustajoki, S., Kauppinen, R., Mustajoki, P., Suomalainen, A.& Peltonen, L. Genome Res. 7, 1054–1060 (1997).

5. Lolle, S. J., Hsu, W. & Pruitt, R. E. Genetics 149, 607–619(1998).

6. Downs, D. M. & Roth, J. R. Genetics 117, 367–380 (1987).7. Ray, A. Nature doi:10.1038/nature04063 (2005).

doi:10.1038/nature04062Reply: Lolle et al. reply to this communication(doi:10.1038/nature04064).

PLANT GENETICS

Hothead healer and extragenomic informationArising from: S. J. Lolle, J. L. Victor, J. M. Young & R. E. Pruitt Nature 434, 505–509 (2005)

HTH ATTCGGCCGTCGTCACACCGChth-4 ATTCGGCCGTtGTCACACCGCRS TCGGCCGTCGTCACA

HTH CGAGTCTCCAGGAACCAACCChth-8 CGAGTCTCCAaGAACCAACCCRS GTCTCCAGGAACCAA

HTH CAGACTGTTGGAATTACAAAGhth-10 CAGACTGTTGaAATTACAAAGRS AGACTGTTGGAATTACAA

ER TATGCTTTCTTAAGCer TATGCTaTCTTAAGCRS TATGCTTTCTTAAGC

Figure 1 | DNA nucleotide sequences of the hth-4,hth-8, hth-10 and er mutants in the region of themutation, compared with wild type. Sequences ofthe mutants are shown in blue, with the mutatednucleotide in lower-case; the corresponding wild-type sequences are in black and the nucleotide at the site of mutation is highlighted in red.Homologous sequences that might cause themutations to revert (RS sequences), obtained byBLAST-searching the Landsberg erecta databasefrom www.arabidopsis.org, are shown in green,with the wild-type nucleotide in red. Reversionfrequency is lower for the hth-4 allele (with 6 RSsequences of 13–15 nucleotides), than for hth-8(with 20 RS of 13–15 nucleotides) and hth-10(with 24 RS and 13–18 nucleotides).

PLANT GENETICS

RNA cache or genome trash?Arising from: S. J. Lolle, J. L. Victor, J. M. Young & R. E. Pruitt Nature 434, 505–509 (2005)

According to classical mendelian genetics,individuals homozygous for an allele alwaysbreed true. Lolle et al.1 report a pattern of non-mendelian inheritance in the hothead (hth)mutant of Arabidopsis thaliana, in which aplant homozygous at a particular locus uponself-crossing produces progeny that are 10%heterozygous; they claim that this is the resultof the emerging allele having been reintro-duced into the chromosome from a cache ofRNA inherited from a previous generation.Here I suggest that these results are equallycompatible with a gene conversion thatoccurred through the use as a template of

DNA fragments that were inherited from aprevious generation and propagated inarchival form in the meristem cells that gener-ate the plant germ lines. This alternative modelis compatible with several important observa-tions by Lolle et al.1.

Such archival forms of DNA have beendescribed previously2. The template DNAcould have originated in the fragmentedgenomes of three of the four haploid femalemeiotic products in the germline of a hetero-zygous plant. Within the ovule of the originalheterozygous plant, the surviving haploidfemale germline cell containing the mutant hth

© 2005 Nature Publishing Group

BRIEF COMMUNICATIONS ARISING NATURE|Vol 437|1 September 2005

E2

allele acquires from the degenerating sisternuclei a collection of chromatin fragments thatare presumably heterochromatinized andsilenced; they might also be covalently modi-fied and therefore hard to detect by Southernblotting or by amplification in the polymerasechain reaction.

I propose that these supernumerary chro-matin fragments propagate within the meri-stem cells of succeeding generations, and so arepresent in a very few cells of the plant; never-theless, they are often present in the germ linesthat are themselves derived from the meristem.Within a second-generation descendant that ishomozygous for a chromosomal allele, suchsupernumerary chromatin fragments mightharbour another allele in a cryptic state. Thesesupernumerary fragments might pair withnormal chromosomes in the male germ linepreferentially, as indicated by Lolle et al.1,where they direct gene conversion of the chro-mosomal alleles. The gene-converted allele,therefore, would reappear as a mendelian fac-tor in the third or a subsequent generation.

This proposal dispenses with the hypothet-ical RNA cache1, for which little evidenceexists. It is consistent with the incidence ofsupernumerary chromosome fragments in

plants, particularly with their known effects on pollen function3, and explains the lack ofsomatic convertant sector in any generation1;it also invokes standard molecular processes ofDNA-directed gene conversion over relativelylong regions of homology. The differences inthe frequency of conversion of the three testedalleles1 may reflect co-conversion polarity4.

My proposal can also explain the curiouslyhigh frequency of allele reappearance in dis-sected embryos, which is roughly twice thatfound in mature plants (it increased from 10%to 20%). Assume that gene conversion producesa heteroduplex DNA that escapes mismatchrepair. If conversion occurs in the haploid gen-erative nucleus of the pollen, the two spermcells will be non-identical at the convertedallele. After double fertilization, the embryoand the endosperm will contain two differentalleles. An embryo dissected from such a seedwill inevitably be associated with endospermcells, and will yield a signal in the polymerasechain reaction that is indistinguishable from aheterozygous embryo, although the embryoitself is homozygous for the allele. As this willoccur as often as a conversion, the frequencyof the total converted allele in dissectedembryos and endosperms together should be

about 20%, an estimate close to the experi-mentally observed rate.

The model proposed here, but not one inwhich gene conversion is directed by RNA5

or by very short, dispersed, repeated DNAsequences6, is easily reconciled with the notionof co-conversion polarity4. It should be possi-ble to test whether co-conversion polarity is afactor in the phenomenon revealed by Lolle etal.1 by producing double- and triple- mutantalleles through exploitation of this effect. Fur-ther investigations into the possibility andnature of DNA archiving in plants and of plantgerm lines should prove interesting.Animesh RayKeck Graduate Institute, Claremont, California91711, USAe-mail: aray@kgi.edu

1. Lolle, S. J., Victor, J. L., Young, J. M. & Pruitt, R. E. Nature434, 505-509 (2005).

2. Downs, D. M. & Roth, J. R. Genetics 117, 367–380 (1987).3. Stebbins, G. L. in Chromosomal Evolution in Higher Plants

67–71 (Addison-Wesley, Reading, Massachusetts, 1971).4. Nicolas, A. & Petes, T. D. Experientia 50, 242–262 (1994).5. Baltimore, D. Cell 40, 481–482 (1985).6. Chaudhury, A. Nature doi:10.1038/nature04062 (2005).

doi:10.1038/nature04063Reply: Lolle et al. reply to this communication(doi:10.1038/nature04064).

PLANT GENETICS

Lolle et al. replyReply to: A. Chaudhury (doi:10.1038/nature04062) and A. Ray (doi:10.1038/nature04063)

Chaudhury1 and Ray2 propose alternativemodels to account for our observed pattern ofnon-mendelian inheritance in the hothead(hth) mutant of Arabidopsis3.

Chaudhury suggests that the informationrequired to restore correct genetic sequencesin hth mutant plants could be stored in shortstretches of nucleotide sequence within thegenome1. Although the sequences required forrestoration are indeed present in the genome,the length of similarity seen in the ‘revertingsequences’ identified by Chaudhury is barelygreater than would be expected from randomchance. An appropriate control for his in silicoexperiment would be to establish how manysimilar sequences (13–18 nucleotides inlength, with a single nucleotide mismatch rel-ative to the sequence in the parent plant) arepresent in the genome that could likewiseintroduce silent nucleotide substitutions intothe hth gene under the same conditions.

Should there be a significant number ofthese sequences (and given that no such silentmutations occur in the corrected alleles3), thenan explanation is needed for why some ofthem are used for correction whereas themajority are not. Even if silent mutation eventsoccur independently of the reversion of thehth mutation, we should still detect them inour small sample of sequenced reverted alleles

owing to the relatively large number of possi-ble silent mutations.

Chaudhury also suggests that the increasedpermeability of mutant hth female gameto-phytes could allow DNA or RNA from thedegenerating non-functional megaspores toenter the functional hth megaspore and then bearchived and carried forward to allow geneconversion in the next generation. This may bea possibility, but our previous work examinedonly changes in the permeability of the extra-cellular cuticle covering the outside of the epi-dermal cell layer4. We have no data concerningincreased cellular permeability in hth mutants.The fact that we see no obvious drop in the rateof reversion over several generations3 is incon-sistent with Chaudhury’s suggestion that thiscould be a second mechanism to bolster therate of reversion for only a single generation.

Ray2 claims that our results could beexplained by the stable inheritance of super-numerary chromosomal fragments that arearchived in a way that makes them inaccessi-ble to DNA hybridization and the polymerasechain reaction. These fragments might also berestricted to meristematic cells and thereforebe present in such low concentrations thatthey are undetectable in a conventional exper-iment. This is an interesting possibility that isconsistent with our observations, but it postu-

lates a novel system of segregation to restrictthe chromosome fragments to what wouldconstitute a hitherto undetected germ line inplants. Considering also Ray’s explanation forthe doubled rate of conversion in embryos, wenote that it would be necessary for all conver-sion events to take place in the generative celland to fail to be corrected by mismatch repair.

In summary, we agree with Ray that there islittle direct evidence to support any given mol-ecular identity for the cryptic templates thatallow genetic restoration in hth mutant plants.We proposed that the templates might be areplicating form of RNA, but the data are alsoconsistent with a form of DNA that is segre-gated into a limited number of cells in theplant or that is not readily detectable by conventional molecular techniques. Thissequence archive (whether DNA or RNA)would therefore require the same basic prop-erties as those we proposed3: it would need tobe replicated, transmitted with high fidelityover several generations, and retain the abilityto restore nuclear DNA sequences.Susan J. Lolle, Jennifer L. Victor,Jessica M. Young, Robert E. PruittDepartment of Botany and Plant Pathology,Purdue University, West Lafayette,Indiana 47907-2054, USAe-mail: pruittr@purdue.edu

1. Chaudhury, A. Nature doi:10.1038/nature04062 (2005). 2. Ray, A. Nature doi:10.1038/nature04063 (2005). 3. Lolle, S. J., Victor, J. L., Young, J. M. & Pruitt, R. E. Nature

434, 505–509 (2005).4. Lolle, S. J., Hsu, W. & Pruitt, R. E. Genetics 149, 607–619

(1998).

doi:10.1038/nature04064

© 2005 Nature Publishing Group