Hothead ( hth/hth ) mutants in Arabidopsis

2. Hothead(hth/hth) mutants in Arabidopsis • All Hothead mutations are recessive – need • both mutant alleles to produce phenotype • All mutant alleles contain single point mutations • which lead to different restriction site digestion patterns • Hothead mutant phenotypes characterized thus far: • Floral organ fusion • Cuticles have increased cellular permeability • Increased rate of chlorophyll extraction due • to possible changes in permeability in cell wall • Pollen hydration on vegetative surfaces

3. Self-fertilized homozygous hothead mutants (hth/hth) revert to heterozygous condition (hth/HTH) at an unusually high rate

4. Molecular genetic analysis of HTH PCR-based assay demonstrating genetic reversion of hth-8. DNA was extracted from mutant and revertant progeny that were derived from the self-fertilization of a homozygous hth-8 parent, amplified with gene specific primers and digested to reveal the presence or absence of a polymorphic restriction site. Controls in the left three lanes show digest patterns derived from wild-type (HTH/HTH), heterozygous (HTH/hth) and homozygous (hth/hth) mutant plants. Photos above mutant and revertant lanes show the hth/hth mutant phenotype and the revertant phenotype.

5. Possible alternative explanations for results • Presence of transposons and repeated sequences • Seed contamination due to WT plants grown in close proximity • Outcrossing of the hth mutant plants with wild-type pollen • High rate of random mutation in this particular region of the genome • Correction of the gene through gene conversion mechanisms

6. Experiment to test seed contamination hypothesis • Individual embryos were dissected out of fruits developing on • selfed homozygous hth/hth mutant plants and genotyped

7. Can pollen derived from hth/hth mutant plants transmit an HTH wild-type allele to progeny? • Homozygous mutant plants (hth/hth) in Ler background were reciprocally • crossed with wild-type Col plants and the embryos were genotyped • Most embryos were heterozygotes as expected • In crosses where the female parent was homozygous mutant hth/hth, • no HTH/HTH embryos were detected • In crosses where the male plant was hth/hth, 8/164 embryos were found • with HTH/HTH genotype • Results indicate there is a bias for these genetic changes to occur in the male • reproductive system

8. Testing for a high rate of random mutations at the HTH locus • 1,226 possible silent nucleotide substitutions in the HTH coding region • Expect to see 49-100 other nucleotide changes on average • in the coding sequence of reverted alleles • Sequenced the complete coding region of the HTH gene after three • independent reversion events in each of the three mutant hth alleles • All sequence of the reverted HTH allele matched the wild-type • sequence exactly indicating that is not random sequence changes

9. Testing gene conversion hypothesis – Genome blot indicates HTH is a single copy gene in Arabidopsis Genome blot showing that HTH is present in a single copy in Col (C), Ler (L) and Ws (W). Genomic DNA was digested as indicated and probed with a labelled fragment of Col DNA corresponding to the HTH coding sequence.

10. Examining the family of Hothead-like genes as a source for gene conversion Figure 2 DNA sequences of HTH and HTL genes around the sites of mutation in hth-4,hth-8 and hth-10. The location of the mutant nucleotide is boxed, and the wild-type, nucleotide at this position is shown in blue; differences from the HTH sequence are shown in red lower-case letters. For hth-4 none of the related sequences contain the wild-type nucleotide at the site of the mutation, for hth-8 all of the other sequences contain the appropriate wild-type nucleotide, and for hth-10 some of the other sequences contain the relevant wild-type nucleotide. In all cases there are sequence differences between the HTL sequences and HTH within a few nucleotides of the site of the mutation.

11. Do DNA sequences changes occur in other places in the genome? • Crossed hth/hth mutants in Ler genetic background with wild-type Col plants • Allowed the F1 hybrids to self-fertilize • Genotyped F2 for several polymorphic markers between Ler and Col

12. Template driven mechanism – RNA cache hypothesis vs. reverting sequence hypothesis Figure 1 | DNA nucleotide sequences of the hth-4, hth-8, hth-10 and er mutants in the region of the mutation, compared with wild type. Sequences of the mutants are shown in blue, with the mutated nucleotide in lower-case; the corresponding wildtype sequences are in black and the nucleotide at the site of mutation is highlighted in red. Homologous sequences that might cause the mutations to revert (RS sequences), obtained by BLAST-searching the Landsberg erecta database from www.arabidopsis.org, are shown in green, with the wild-type nucleotide in red. Reversion frequency is lower for the hth-4 allele (with 6 RS sequences 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).