Plant Molecular Biology 36: 811–820, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
AIR synthetase in cowpea nodules: a single gene product targeted to two
Penelope M.C. Smith
, Anthea J. Mann, Danica E. Goggin and Craig A. Atkins
Department of Botany, The University of Western Australia, Nedlands, Western Australia, 6907, Australia
author for correspondence)
Received 28 February 1997; accepted in revised form 14 November 1997
Key words: gene expression, nodules, phosphoribosyl aminoimidazole synthetase, purine biosynthesis, N assimil-
AcDNA(VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the ﬁfth enzyme of the de novo
purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid
protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has signiﬁcant homology
with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea
nodules . A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit
sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although
the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial
cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests
that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to
the two organelles.VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the
gene is active before nitrogen ﬁxation begins. These results suggest that products of nitrogen ﬁxation do not play
a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant
protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular
size. Both formsare present in mitochondria,although the larger protein is more abundant. Only the smaller protein
was detected in plastids.
De novo synthesis of purine is the major pathway for
assimilation of ﬁxed nitrogen in tropical legumes such
and other members of the tribe Phaseoleae. The nuc-
leotide product of purine biosynthesis, inosine mono-
phospate (IMP), is hydrolyzed and partially oxidized
in the infected cells and then transported to the adja-
cent uninfected cells where it is further oxidized to the
ureides: allantoin and allantoic acid. These are expor-
ted from the nodules in xylem and are the principal
source of N for amino acid and protein synthesis in
Thenucleotidesequencedata reportedwill appear intheEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number U30895.
the legume . As a consequence of the high rates of
nitrogen assimilation in nodules, the rates of purine
synthesis in infected cells are much greater than those
in other plant tissues [2, 3].
Not all legumes form purines and ureides as
products of N
ﬁxation in nodules. Temperate species
like peas (Pisum sativum), lupins (Lupinus angustifo-
lius) and clover (Trifolium spp.) form asparagine .
Furthermore, when non-nodulated and supplied with
reduced N (e.g. NO
), cowpeas and soybeans form
asparagine rather than ureides and the level of expres-
sion of the purine pathway in roots is low [1, 6]. Thus
enhanced expression of the purine pathway is nodule-
speciﬁc but is restricted to certain symbioses.
The pathway for de novo synthesis of purines
involves ten enzymatic steps to produce IMP from