Plant Molecular Biology 39: 395–405, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Complexity and expression of the glutamine synthetase multigene family
in the amphidiploid crop Brassica napus
, Gerald Schock, Martin Trischler, Kirstin Kosemund and Aloysius Wild
Institut für Allgemeine Botanik, Johannes Gutenberg-Universität Mainz, 55099 Mainz, Germany (
Received 6 November 1997; accepted in revised form 23 July 1998
Key words: amphidiploid genome structure, gene expression, glutamine synthetase, multigene family, nitrogen
assimilation, oilseed rape
In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and
B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families
of the A and C genome types encoding a certain protein are now combined in one plant genome.
In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six dif-
ferent cDNA sequences were isolated from leaf and root speciﬁc libraries. One sequence pair (BnGSL1/BnGSL2)
was characterized by the presence of amino-terminal transit peptides, a typical feature of all nuclear encoded
chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high
homology between each other were found in a root speciﬁc cDNA library and represent protein subunits for
cytosolic glutamine synthetase isoforms.
Comparative PCR ampliﬁcations of genomic DNA isolated from B. napus, B. campestris and B. oleracea
followed by sequence–speciﬁc restriction analyses of the PCR products permitted the assignment of the cDNA se-
quences to either the A genome type (BnGSL1/BnGSR1-1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-
2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously
in oilseed rape. This result was also conﬁrmed by RFLP (restriction fragment length polymorphism) analysis of
In addition, the different GS genes showed tissue speciﬁc expression patterns which are correlated with the state
of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a
marked increase of expression could be observed after the onset of leaf senescence.
The ATP-dependent synthesis of glutamine, a most
important form of N storage and of N transport in
the plant nitrogen metabolism, is catalysed by the
enzyme glutamine synthetase (GS; EC 22.214.171.124). In col-
laboration with glutamate synthase (NADH-GOGAT;
EC 126.96.36.199 or FD-GOGAT; EC 188.8.131.52), another
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers X72751 (BnGSL1), X76736 (BnGSR1-1),
X82997 (BnGSR2-1), Y12458 (BnGSL2), Y12459 (BnGSR1-2)
and Y12460 (BnGSR2-2).
important carrier amino acid, glutamate, is formed
. This GS/GOGAT cycle is responsible for the
(re)assimilation of ammonia generated by nitrate re-
duction, symbiotic nitrogen ﬁxation, and photorespi-
ration or released during catabolic reactions like leaf
senescence and seed germination [22, 29].
After the assimilation of ammonia into glutamine
and glutamate, these two amino acids are used as
nitrogen donors in the biosynthesis of aspartate and
asparagine. Two major enzymes are involved in this
downstream metabolism. The shift of assimilated ni-
trogen into aspartate or asparagine is catalyzed by
the enzymes aspartate aminotransferase (AspAT; EC