Plant Molecular Biology 37: 131–140, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Molecular cloning and characterization of an inorganic pyrophosphatase
, Sjoukje Heimovaara-Dijkstra, Jan W. Kijne and Mei Wang
Center for Phytotechnology,Leiden University/Netherlands Organization for Applied Scientiﬁc Research,
Wassenaarseweg 64, 2333 AL Leiden, Netherlands (
author for correspondence)
Received 6 November 1997; accepted in revised form 23 December 1997
Key words: dormancy,germination, Hordeum, inorganic pyrophosphatase
A cDNA clone with sequence homology to soluble inorganic pyrophosphatase(IPPase) was isolated from a library
of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli,and
showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active
tissue such as root, shoot, embryoand aleurone. During imbibition, a continuousincrease of the steady state mRNA
level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production
declined, after an initial increase. With isolated dormantand nondormantembryos, addition of recombinantIPPase,
produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate(PPi), substrate
for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.
Development and germination of a barley grain
requires a large metabolic activity. In the germinat-
ing embryo, degradation of reserves is followed by
uptake of nutrients from the endosperm. ATP hydro-
lysis provides the driving force for many metabolic
reactions. For example, one of the processes in the
germinating embryo is the degradation of fatty acids
-oxidation of fatty acids is coupled to the
hydrolysis of ATP into AMP and pyrophosphate (PPi)
ofPPi. The hydrolysisof PPi intoinorganicorthophos-
thermodynamically irreversible .
Inorganic pyrophosphatases are able to catalyse
the hydrolysis of PPi into two molecules of ortho-
phosphate. These enzymes are ubiquitously present
in nature. Their important role in metabolism is illus-
trated by the observation that expression of soluble
inorganicpyrophosphatase(IPPase) of bacterial origin
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AF009675.
in the cytosol of transgenic tobacco and potato plants
leads to a signiﬁcant alteration in metabolism, growth
and development [9, 13, 20]. With plants, the cloning
of soluble IPPase was reported for Arabidopsis and
potato [4, 11].
We are interested in the physiology of dormancy
in barley. We screened an expression library of devel-
oping barley grains with an antibody that was raised
against a putative ABA-binding protein fraction [24,
26]. One of the two positive clones found showed
homology to genes encoding soluble inorganic pyro-
phosphatase (IPPase). Considering the origin of this
cloneand the roleof IPPaseinmetabolism, wedecided
to test the working hypothesis that this IPPase is
involved in regulation of germination of barley grains.
Since ABA plays a key role in germination as well
as in the induction and maintenance of dormancy of
seeds [1, 10, 21, 23, 24, 28, 29], we also studied the
interactions between IPPase, PPi and ABA. Our res-
ults suggest that PPi elevatesthe extra-embryonallevel
of ABA, thereby inhibiting germination. By removing
PPi, IPPase could indirectly stimulate germination.