Plant Molecular Biology 51: 523–531, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Differences in the processing of DNA ends in Arabidopsis thaliana and
tobacco: possible implications for genome evolution
Nadiya Orel and Holger Puchta
Institut für Pﬂanzengenetik und Kulturpﬂanzenforschung (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany
(*author for correspondence; e-mail firstname.lastname@example.org)
Received 29 November 2001; accepted in revised form 8 August 2002
Key words: C value paradox, deletion, DNA recombination, NHEJ, plant genome size
Surprising species-speciﬁc differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks
(DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum.In
Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish
the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic
transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of
the nature of the ends (blunt, 5
overhangs), linearized full-length DNA molecules were, on average, more
stable in tobacco than in Arabidopsis. The relative expression of a β-glucuronidase gene encoded by the plasmid
was similar in both plant species when the break was distant from the marker gene. However, if a DSB was
introduced between the promoter and the open reading frame of the marker, transient expression was halved in
Arabidopsis as compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than
in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or
both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the
Arabidopsis genome is more than twenty times smaller than the tobacco genome.
The elucidation of the complete sequence of the Ara-
bidopsis genome revealed multiple sequence duplica-
tions, although the genome consists of only 125 Mb
and is thus one of the smallest plant genomes known.
This hints to the possibility of a genome duplication
in the evolution of Arabidopsis (Arabidopsis Genome
Initiative). Thus, ample amounts of DNA must have
been lost after the postulated duplication event, result-
ing in the small size of today’s Arabidopsis genome.
Recently, programmed sequence elimination in the
ﬁrst generations after allopolypolydization of wheat
could be demonstrated (Ozkan et al., 2001; Shakled
et al., 2001), but further DNA elimination over longer
evolutionary time periods has to be postulated to ex-
plain the amount of sequence loss (Petrov, 2001). Indi-
cations for species-speciﬁc deletions were ﬁrst found
in insects. Studies on non-coding retrotransposon-like
sequences of insects indicated considerable DNA loss
from the Drosophila genome (Petrov, 1996). Indeed,
the rate of deletions differed drastically between dif-
ferent insect genomes. Cricket, which has a 11-fold
larger genome than Drosophila has a 40-fold lower
rate of DNA loss than Drosophila. This applies for the
number as well as the size of the deletions. Based on
this ﬁnding, an inverse correlation between genome
size and deletions size was postulated (Petrov et al.
But, what kind of process might be responsible
for these deletions? Deletions may occur by different
mechanisms: by replication slippage (as suggested by
Capy, 2000), by unequal crossover (as suggested by
Smith, 1976) or by double-strand break (DSB) repair.
Recently we compared DSB-induced deletions that re-
sult in a loss of function of a chromosomal marker
gene in two related dicotyledonous plant species Ara-
bidopsis and tobacco (Kirik et al., 2000), which differ