Plant Molecular Biology 41: 551–561, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
The DapA gene encoding the lysine biosynthetic enzyme
dihydrodipicolinate synthase from Coix lacryma-jobi: cloning,
characterization, and expression analysis
Ricardo Augusto Dante
, Germano Cord Neto
Departamento de Gen´etica e Evolução, Instituto de Biologia, and
Centro de Biologia Molecular e Engenharia
Gen´etica, Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil (
author for correspondence;
Received 26 April 1999; accepted in revised form 2 September 1999
Key words: Coix lacryma jobi, dihydrodipicolinate synthase, GCN4, lysine, maize, opaque2
Dihydrodipicolinate synthase (DHPS) is the main enzyme of a speciﬁc branch of the aspartate pathway leading to
lysine biosynthesis in higher plants. We have cloned and characterized the DHPS-encoding DapA gene from the
maize-related grass Coix lacryma-jobi.TheDapA open reading frame is interrupted by two introns and encodes
the 326 amino acid-long Coix DHPS protein, which is 95% identical to the maize DHPS protein. Coix DNA gel
blot analysis with maize DHPS cDNA as a probe showed a single strongly hybridizing band along with faint bands.
RNA gel blot analysis showed that DHPS transcripts are present in coleoptiles, embryos, endosperms, and roots but
are almost undetectable in blades of young leaves of both Coix and maize. The 5
-ﬂanking region of the DapA gene
contains a TGACTC GCN4-like element located 372 bp upstream the putative translation start codon. Steady-state
levels of DHPS mRNA were slightly reduced in the endosperms and embryos of the maize lysine-rich opaque2
mutants when compared with those in normal kernels. Selective binding assay with the maize Opaque2 protein
(O2) showed that the GCN4-like element is not an O2 binding site, suggesting that the DHPS gene is not under the
control of O2.
In many bacteria and in higher plants, lysine, threo-
nine, methionine and isoleucine are synthesized by the
aspartate family biosynthetic pathway (Galili, 1995;
Azevedo et al., 1997). Two enzymes of the path-
way, aspartate kinase (AK) and dihydrodipicolinate
synthase (DHPS), are feedback-inhibited by lysine.
DHPS is the ﬁrst enzyme of the branch that leads ex-
clusively to lysine biosynthesis (Bryan, 1990). Various
reports have demonstrated that DHPS activity is the
key regulatory step for lysine biosynthesis in plants
(Galili, 1995; Azevedo et al., 1997). The enzyme is
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Database under the
accession number U61730.
particularly sensitive to lysine feedback inhibition (I
of 10–50 µM, in contrast to 100–700 µM for AK;
Galili, 1995). Mutant and transgenic plants expressing
forms of DHPS less sensitive to lysine feedback inhi-
bition accumulate free lysine (Negrutiu et al., 1984;
Glassmann, 1992; Perl et al., 1992; Shaul and Galili,
1992; Falco et al., 1995; Brinck-Pedersen et al., 1996;
Tzchori et al., 1996). Moreover, expression of both
insensitive DHPS and AK leads to higher lysine accu-
mulation at the expense of threonine (Frankard et al.,
1992; Shaul and Galili, 1993). Furthermore, the posi-
tive correlations seen between DHPS activity or DHPS
protein level and free lysine content suggest that the
amount of the enzyme may inﬂuence lysine accumu-
lation (Perl et al., 1992; Shaul and Galili, 1993; Falco
et al., 1995).