Supporting Figures S1 – S3
Supporting Table S1
Figure S1. Characterisation of Atmhf1-1 and Atmhf1-2 mutant lines. (a) Detailed analysis of
T-DNA insertion sites in both mhf1 mutant lines. Genomic sequences are shown in bold,
additional insertions in italic, deletions are abbreviated as Del and their specific lengths are
given. (b) MHF1 gene expression across and downstream of T-DNA insertion in mhf1-1 and
mhf1-2 mutant lines in relation to the measured gene expression in WT plants. For mhf1-1
downstream of the insertion site an expression level increased compared to WT plants was
observed. In mhf1-2, downstream of the T-DNA insertion an expression level similar to that of
the WT was detected. Across T-DNA insertion, no or a very low expression was determined in
both mhf1 mutant lines. Since there are only very few bp of transcript 5’ of both insertions,
expression levels upstream of the T-DNA insertion site could not be measured.
Figure S2. Relative fresh weight of Atmhf1-1 and Atmhf1-2 after the treatment with
bleomycin (bleo) (a), hydroxyurea (HU) (b) or methyl methanesulfonate (MMS) (c),
respectively. The relative fresh weights of the tested lines were calculated as fresh weight of
the line after genotoxin treatment in relation to the fresh weight of untreated control plants of the
same line. Each assay was performed at least three times to calculate the standard deviations
(error bars). The induction of genotoxic stress with neither of those genotoxins led to a
difference in the relative fresh weight compared to WT plantlets.
Figure S3. Relative fresh weights of different double mutants after cis-Platin treatment.
The relative fresh weights of the tested lines were calculated as fresh weight of the line after
genotoxin treatment in relation to the fresh weight of untreated control plants of the same line.
Each assay was performed at least three times to calculate the standard deviations (error bars).
(a) The relative fresh weight of the mhf1-1 recq4A-4 double mutant displayed a reduction in
fresh weight after 5 and 10 µM cis-Platin treatment similar to that observed with the recq4A-4
single mutant. (b) The mhf1-1 mus81-1 double mutant showed no significant difference in terms
of sensitivity to 5 and 10 µM cis-Platin compared to the mus81-1 single mutant. (c) As the
induction of genotoxic stress with 5 and 10 µM cis-Platin led to a slightly reduced but not
significantly reduced fresh weight, also lower concentrations of cis-Platin were tested. After 1.5
and 2.5 µM cis-Platin treatment, the mhf1-1 mus81-1 revealed no further reduction in fresh
weight compared to the mus81-1 single mutant, as well. (d) The mhf1-1 fancm-1 double mutant
displayed similar fresh weights in comparison to both single mutants and WT plants.
Table S1. Segregation analysis of the fancm-1 mus81-1 double mutant.
To test if the double mutant of fancm-1 and mus81-1 is viable, we grew progeny from a line
homozygous for mus81-1 and segregating for fancm-1 on solid GM plates and tested the
segregation of seedling viability by a χ2 test with 1 degree of freedom. One would expect the
classical distribution of 1/4 of fancm-1-/- mus81-1-/- plants, 2/4 of fancm-1-/+ mus81-1-/- plants and
1/4 of fancm-1+/+ mus81-1-/- plants. If the fancm-1 mus81-1 double mutant is not viable, 1/4 of
the progeny should therefore not be able to grow. In our tests, in 5 biological replicates of this
segregation analysis the numbers of viable and dead seedlings followed a 3:1 segregation since
the χ2 values are well below the critical value of 3.84. Furthermore, genotyping of the population
of growing seedlings resulted in the identification of only plants that were heterozygous for the
fancm-1 mutation (fancm-1
-/+)
or WT (fancm-1
+/+).
Therefore, we conclude that the progeny
that were homozygous for both fancm-1 and mus81-1 died very early during vegetative growth.