Plant Molecular Biology 49: 171–186, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Characterization of a tissue-speciﬁc and developmentally regulated
β-1,3-glucanase gene in pea (Pisum sativum)
, Christine Rochat
, Sylvie Wuill
eme and Jean-Pierre Boutin
Laboratoire de Biologie des Semences, INRA, RD 10, 78026 Versailles cedex, France (*author for correspondence;
current address: IACR Rothamsted, Dept. of Biochemistry & Physiology,
Harpenden, Herts. AL5 2JQ, UK
Received 20 February 2001; accepted in revised form 19 October 2001
Key words: callose, expression analysis, β-1,3-glucanase, Pisum sativum, promoter analysis, seed development,
As part of a search for seed coat-speciﬁc expressed genes in Pisum sativum cv. Finale by PCR-based methods, we
identiﬁed and isolated a cDNA encoding a β-1,3-glucanase, designated PsGNS2. The deduced peptide sequence of
PsGNS2 is similar to a subfamily of β-1,3-glucanases, which is characterized by the presence of a long amino acid
extension at the C-terminal end compared to the other β-1,3-glucanases. PsGNS2 is expressed in young ﬂowers and
in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is
not inducible by environmental stress or in response to fungal infection. In developing pea ﬂowers the transcript is
detectable in all four whirls. In the seed coat the expression is temporally and spatially regulated. High abundance
of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the
mid seed-ﬁlling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip
of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo
nutrition. This tissue showed also less callose deposits than the other ones. The 5
genomic region of PsGNS2
was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-speciﬁc expression.
Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed.
Abbreviations: DIG, digoxigenin; GUS, β-glucuronidase; RT-PCR, reverse transcriptase-polymerase chain
Plant β-1,3-glucanases represent a highly diverse fam-
ily of hydrolytic enzymes. This diversity is explained
by differences in the regulation of their gene expres-
sion, in the chemical and structural feature of their
protein and also in their cellular localization. Their ex-
clusive substrates are 1,3-glucans such as callose and
laminarin found in the cell walls of plants and fungi.
They are generally induced in response to pathogen
attack or environmental stress (Kauffman et al., 1987;
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers AJ251646 and AJ251199.
Simmons et al., 1992; Cruz-Ortega et al., 1995). A
plant defence-related biological function for these hy-
drolytic enzymes has been suggested based on their
co-ordinated induction, together with chitinases, and
their inhibitory effect on in vitro growth and sporu-
lation of pathogenic fungi (Jach et al., 1995; Mauch
et al., 1988a, b; Vogeli et al., 1988). Another pro-
posed defence role of β-1,3-glucanases is the release
of elicitors from pathogen cell walls for the induction
of the defence response (Keen and Yoshikawa, 1983).
Nevertheless, tissue speciﬁcity and developmentally
regulated non-pathogen-induced expression of β-1,3-
glucanase genes have also been reported (Lotan et al.,
1989; Memelink et al., 1990; Hird et al., 1993).