Germin-like protein gene family of a moss, Physcomitrella patens,
phylogenetically falls into two characteristic new clades
Masaru Nakata*, Yayoi Watanabe, Yoko Sakurai, Yuka Hashimoto, Masahiro
Matsuzaki, Yohsuke Takahashi and Toshio Satoh
Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1,
Higashi-Hiroshima, 739-8526 Japan (*author for correspondence; e-mail firstname.lastname@example.org)
Received 31 July 2004; accepted in revised form 20 September 2004
Key words: bryophyte subfamily, germin-like protein, phylogenetic analysis, Physcomitrella patens,
We identiﬁed 77 EST clones encoding germin-like proteins (GLPs) from a moss, Physcomitrella patens in a
database search. These Physcomitrella GLPs (PpGLPs) were separated into seven groups based on DNA
sequence homology. Phylogenetic analysis showed that these groups were divided into two novel clades
clearly distinguishable from higher plant germins and GLPs, named bryophyte subfamilies 1 and 2.
PpGLPs belonging to bryophyte subfamilies 1 lacked two cysteines at the conserved positions observed in
higher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2 contained two cysteines as
observed in higher plant germins and GLPs. In bryophyte subfamily 1, 12 amino acids, in which one of two
cysteines is included, were deleted between boxes A and B. Further, we determined the genomic structure of
all of seven PpGLP genes. The sequences of PpGLPs of bryophyte subfamily 1 contained one or two
introns, whereas those of bryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, a
liverwort GLP from Marchantia polymorpha, and two moss GLPs from Barbula unguiculata and Ceratodon
purpureus also fell into bryophyte subfamily 1 and bryophyte subfamily 2, respectively. No higher plant
germins and GLPs were grouped into the bryophyte subfamilies 1 and 2 by our analysis. Moreover, we
revealed that PpGLP6 had manganese-containing extracellular superoxide dismutase activity. These results
indicated that bryophyte possess characteristic GLPs, which phylogenetically are clearly distinguishable
from higher plant GLPs.
Abbreviations: EST, expressed sequence tag; GLP, germin-like protein; H
, hydrogen peroxide; OXO,
oxalate oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase
Germin is a protein that was ﬁrst isolated in
association with wheat germination (Thompson
and Lane, 1980). This protein is localized to the
cell wall and is a water-soluble glycoprotein with
oxalate oxidase (OXO) activity that converts
oxalate to hydrogen peroxide (Lane et al., 1993).
It forms an oligomer that is highly resistant to
protease and heat treatment (Lane, 1994; Dunwell
et al., 2000). Proteins with sequence similarity to
germins have been identiﬁed in various land plants
and are designated germin-like proteins (GLPs).
Germin and GLPs are composed of three highly
conserved oligopeptides that are called germin
boxes A, B, and C (Bernier and Berna, 2001). The
region from boxes B to C, which is called the cu-
pin domain, contains three histidines and a glu-
tamate involved in metal binding and forms the
core beta-barrel structure. Two cysteines that
stabilize the N-terminal structure by forming a
disulﬁde bond and an N-glycosylation site (NXS/
Plant Molecular Biology 56: 381–395, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.