Plant Molecular Biology 39: 979–990, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Selection of Arabidopsis mutants overexpressing genes driven by the
promoter of an auxin-inducible glutathione S-transferase gene
Dianne A.M. van der Kop
, Monique Schuyer, Johan E. Pinas, Bert J. van der Zaal and Paul J.J.
Institute of Molecular Plant Sciences and RUL-TNO Centre for Phytotechnology, Clusius Laboratory, Leiden
University, Wassenaarseweg 64, 2333 AL Leiden, Netherlands;
Present address: ATO-DLO, Bornsesteeg 58,
6708 PD Wageningen, Netherlands
Received 24 February 1998; accepted in revised form 23 November 1998
Key words: Arabidopsis thaliana, glutathione S-transferase, auxin-responsive gene expression, developmental
mutants, GST, gup mutant
Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-
inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well
as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in
which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related,
endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of
the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic
cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.
Auxin, the ﬁrst plant hormone to be discovered, is
known to play an essential role in many processes
in plants. Many physiological studies have illustrated
that auxins inﬂuence plant growth and development
by being essential for cell division and cell elongation
and by determining apical dominance, root architec-
ture and tropic behaviour. Despite the importance of
the hormone, only little is known about the molec-
ular mechanism of auxin action. Auxins are thought
to bind to receptors which, as a result of this bind-
ing, transduce signals that regulate gene expression.
For this reason, proteins that are able to bind auxins
have been isolated and are being studied (reviewed
by Jones and Prasad  and Napier and Venis ).
Also, auxin-regulated genes have been isolated and
characterized. While promoter analysis of these genes
has given some ideas about the cis-acting elements in-
volved in the regulation by auxins, the function of a
large part of these genes is still unknown (reviewed by
Hagen  and Sitbon and Perrot-Rechenmann ).
Besides approaches mentioned above, genetics
has been used to obtain insight into the process of
auxin signal transduction. Since phenotypes speciﬁ-
cally linked to auxin had not been identiﬁed, initial
focus was on the isolation of auxin-resistant mutants
(reviewed by Hobbie & Estelle  and Leyser ).
Many of these mutants turned out to be resistant
to other phytohormones such as ethylene, cytokinin
and abscisic acid as well. They often show a dwarf
phenotype and have agravitropic roots. The auxin
biosynthesis genes that are present in the T-DNA of
Agrobacterium tumefaciens have also been introduced
into plants to study the effects of internal auxin over-
production [26, 37, 39]. Arabidopsis thaliana lines
with such transgenes showed a number of morpho-
logical alterations as compared to the wild type: more
pronounced elongation of the hypocotyl and the peti-
oles, epinastic leaves and cotyledons and extensive
root growth (van der Graaff and Hooykaas, unpub-
lished). Recently, arabidopsis mutants were isolated
that showed an extreme root proliferation. These mu-