Complete sequence of the hornwort
(Anthoceros formosae) chloroplast genome: Phylogenety
of early land plants inferred from chloroplast genomes
Masanori Kugita, Takeshi Fujikawa, Akira Kaneko, Yuhei Yamamoto,
Yuko Takeya, and Koichi Yoshinaga
Faculty of Science, Shizuoka University
The nucleotide sequence of the chloroplast DNA from hornwort
(Anthoceros formosae) has almost been completed (Fig.1).
The circular double-stranded DNA of 157 kilo base-pairs (bp)
contained a pair of inverted repeat regions (IRA and IRB) with
15,744 bp each, and a small and a large single copy region (SSC
and LSC) of 20 and 106 kb, respectively. They contained 109 genes
(74 peptide-encoding genes, 31 tRNA and four rRNA genes) and
11 open reading frames (ORFs), which were almost identical with
the other chloroplast genomes of green plants.
Fig.1 Genetic circle map of Anthoceros formosae chloroplast
genome. Genes shown inside the map are transcribed clockwise,
and those outside are transcribed anticlockwise. asterisks denote
split genes. LSC, large single copy region; IR, inverted repeat;
SSC, small single copy region
The differences were found in (1) larger IR than that of liverwort
chloroplast DNA and contained excess genes, ndhB, rps7, and
rps12 with compared to that of liverwort. (2) an intron in
rrn23 in the first discovery in land plants. Two introns
were also found in ORF167, though liverwort contained only one.
(3) rps16 was detected, which was absent in all the land
plants chloroplast except tobacco. On the other hand, no homologs
to ORF42a, ORF135 of liverwort was found.
A large number of RNA editing sites was found in hornwort chloroplast
including U to C conversion as well as C to U. The 286 sites
in 44 transcripts analyzed until now, revealed the creation of
initiation and termination codons, and the conversion of nonsense
codon to sense codon.
Here we report that phylogenetic relationships among early land
plants inferred from these molecular structure in chloroplast
genomes. The phylogenies were analyzed with neighbor-joining
method (NJ) based on 5,194 amino acid sites of 28 proteins encoded
in the plastid genomes (Fig.2).
Fig.2 Plastid phylogeny inferred from chloroplast proteins.
Phylogeny analyzed with neighbor-joining method (NJ) based on
5,194 amino acids of 28 proteins. Bootstrap values with 1,000
replicates are indicated above each branch. The tree was rooted
using Mesostigma viride as an outgroup. Genes used for
the analysis are atpB, atpE, petA, petB, petD, psaC, psaI,
psaJ, psbB, psbE, psbF, psbH, psbJ, psbL, psbM, psbN, psbT, rbcL,
rpl14, rpl20, rpl32, rpoA, rpoB, rps3, rps7, rps11,rps18, rps19.
Mechanically-induced
accumulation response in bryophyte cells
Yoshikatsu Sato1, Akeo Kasota1 and Masamitsu Wada1, 2
1Department of Biological Sciences, Graduate School of Science,
Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji, Tokyo
192-0397, Japan
2National Institute for Basic Biology, Myodaiji, Okazaki 444-8585,
Japan.
Chloroplast movement has been studied in many plants but mainly
as a model system for light signaling. However, we recently showed
that the avoidance response of chloroplasts is induced by mechanical
stimulation in fern protonemal cells. Here we report the discovery
of mechanically induced accumulation response of chloroplasts
in moss Physcomitrella patens, Ceratodon purpureus
and liverwort Marchantia polymorpha. When mechanical stimulation
was directly applied with a capillary to a part of a cell, chloroplasts
moved towards and accumulated at the pressed site within 30 min
after the onset of stimulation in all species tested. In red
light grown protonemata of P. patens, the accumulation movement of chloroplasts was inhibited by 10 µM Cremart but not by 0.1 mM cytochalasin B. To know the contributions of external Ca2+ to the response, we examined the effects of stretch-activated ion channel inhibitor gadolinium, at 100 µM and potent calcium channel inhibitor lanthanum at 1 mM on the accumulation movement. Mechano-relocation of chloroplasts was abolished by these drugs, but no effect was observed in photo-relocation of chloroplasts. These results suggest that influx of external Ca2+ through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motile system is dependent on microtubule.
ULTRASTRUCTURE OF P.
PATENS TIP CAULONEMATA CELLS: UNTREATED, COLD GROWN, SEVERED,
ORYZALIN TREATED, UV-A TREATED
Edward B. Tucker1, Vinoud Sookhdeo1, and Lucy Yin2
1Natural Sciences, Baruch College, CUNY, New York, NY and
2Biology Department, University of Massachusetts, Amherst, MA.
The apical cells of P. patens caulonemata are tip growing
and in the dark they are negatively gravitropic. Our data indicated
that sedimentation of chloro-amyloplasts preceded gravi-curvature,
similar to that previously reported for Ceratodon (Walker
and Sack, 1990; Planta 181: 71-77). Gravi-curvature occurred
if cells were grown at 5o C, and was temporarily inhibited if
cells were either treated with oryzalin or exposed to a flash
of UV-A light. Gravi-curvature did not occur if cells were severed
from the sub-apical cells.
Cells were grown on agar plates, wrapped in foil and placed vertically.
They were not gravi-stimulated. Treatments were: untreated, grown
at 5oC, severed, treated for 5 min with 0.1% oryzalin, or exposed
to UV-A for 10s. Cells were fixed in the dark with 4% paraformaldehyde,
2% glutaraldehyde in 100 mM PIPES pH 6.8 and post-fixed with
2% OSO4 plus 0.8% potassium ferricyanide. Tissue was en bloc
stained with 2% uranyl acetate and embedded in Spurr resin. Sections
were post stained with lead citrate. The ultrastructure of the
apical cell demonstrated organelle domains: an apical ER zone,
a chloro-amyloplast free zone, a chloro-amyloplast zone, a nuclear
zone, and a vacuolar zone. Changes in organelle ultrastructure
and distribution were observed on electron micrographs of the
treated cells. For example, in untreated cells chloro-amyloplasts
aligned in rows seemingly outlined by endoplasmic reticulum.
In cold grown cells chloro-amyloplasts were larger while in severed
cells they were smaller. Chloro-amyloplasts of oryzalin treated
cells accumulated in the tip region. With all of these treatments
the normal alignment of chloro-amyloplasts was altered. Supported
by NASA grant NAG5-3743.
CYTOKININ INDUCED THE
PRODUCTION OF POSITIVELY GRAVI-RESPONDING CAULONEMA IN P.
PATENS
Edward B. Tucker1, Celia D. Knight2 and David, J. Cove2
1Natural Sciences, Baruch College, CUNY, New York, NY, USA
and 2Centre for Plant Sciences, LIBA, University of Leeds, Leeds,
UK.
The mechanism by which plants respond to gravity remains elusive.
While auxin transport and distribution plays the central role
in seed plants, this has not been shown for cryptograms. Mosses
are negatively gravitropic and gtrC mutants have been
produced that are positively gravitropic (Knight et al., 1991,
Mol. Gen. Genetics 230, 12-16). Changing the nitrogen source
or the temperature can change the polarity of gravi-curvature
in some gtr mutants (Knight and Cove, 1989, Env. And Exptl
Bot 29, 57-70). We now report that wild type P. patens,
when grown on media containing BA produced massive numbers of
caulonemata that were positively gravitropic. When the moss colony
was exposed to light, buds were produced on these caulonemata.
The production of positively gravitropic caulonemata was not
altered when moss was grown in media containing NAA as well as
BA. P. patens mutants, that were believed to be deficient
in cytokinin metabolism or uptake were insensitive to BA and
would not produce positively responding caulonema. In gtrC
mutants, BA induced buds and inhibited caulonemata numbers and
growth, but it did not change the polarity of caulonemata. Supported
by PSC-CUNY grant and NASA grant NAG5-3743 to EBT and BBSRC support
of Physcomitrella EST Programme to CDK and DJC.
Ca2+-pumping ATPases
in plants:
functional analysis by targeted disruption in Physcomitrella
patens
Alexander Faltusz, Hauke Holtorf and Ralf Reski
Plant Biotechnology, Freiburg University, Schaenzlestrasse
1, 79104 Freiburg, Germany www.plant-biotech.net
Diverse developmental programs in plants are known to be mediated
by different signal transduction pathways.
Ca2+-pumping ATPases are involved in the calcium homeostasis
in bacteria, animals, fungi and plants. Their task is the maintenance
of low concentrations of calcium in the cell to ensure normal
conditions for signal transduction.
All Ca2+-pumps belong to the superfamily of P-type ATPases, named
after their common phosphorylation site motif. They can be subdivided
into two groups, IIA and IIB. In plants the latter shows an N-terminal
calmodulin binding domain which mediates autoinhibition (1).
These pumps have been described in several plants e.g. Arabidopsis,
rice and cauliflower.
The objective of this project is to better understand the role
of Ca2+-pumping ATPases in plants. Based on the high rate of
homologous recombination events in Physcomitrella (2)
we aim to directly assign a function to one member of the IIB-type
Ca2+-ATPases by targeted disruption of the corresponding gene.
We isolated the full-length sequence (3900 bp) of the Physcomitrella Ca2+-ATPase gene D25. DatabaseÅ@searches show 45-55% protein identity with Ca2+-pumps in other plants. Judging from the conserved regions, D25 can clearly be classified as a P-type IIB pump. Transformation of moss protoplasts with a disruption construct resulted in five knockout mutants. These will be analysed further with respect to the influence of different ion concentrations on their phenotype.
References:
1) Geisler M., Axelsen K. B., Harper J. F., Palmgren M. G.
(2000). Molecular aspects of higher plant P-type Ca2+-ATPases.
Biochim Biophys Acta; 1465 (1-2):52-78.
2) Girke T., Schmidt H., Zähringer U., Reski R., Heinz E. (1998). Identification of a novel D6-acyl-group desaturase by targeted gene disruption in Physcomitrella patens. Plant J;
15(1), 39-48.
Acknowledgements:
This work is made possible by the DFG Graduiertenkolleg ÒMolecular Mechanisms of Plant DevelopmentÓ at Freiburg University.
The Physcomitrella
patens EST (PEP) programme
Celia Knight, Andrew Cuming, David Cove, Beverley Merry, Honglin
Rong, Martin Lomas, David Westheada, Ralph Quatranob
Centre for Plant Science (LIBA); University of Leeds, Leeds
LS2 9JT, UK;
aSchool of Biochemistry and Molecular Biology, University of
Leeds, Leeds LS2 9JT, UK; bDept. of Biology, Washington University,
St Louis, US
PEP is a UK (BBSRC) – funded programme to sequence 30,000
ESTs from Physcomitrella patens for direct submission
to the public gene database GenBank. This programme builds on
a research agreement between the University of Leeds (UK) and
Washington University at St Louis (USA). The remit of the programme
is to generate the sequences for identification of homologues
of higher plant genes in moss. Transformation vectors can then
be constructed for gene targeting in order to determine gene
function. This approach exploits the efficient homologous recombination
system of Physcomitrella which is unique among plants and offers the potential for elucidating novel gene functions as well as the sophistication of allele replacement ( a term we call Ògene surgeryÓ).
PEP provides training courses for the culture and manipulation
of Physcomitrella as well as a service for generating moss transgenics.
The programme is publicised through its web-site (http//:www.moss.leeds.ac.uk)
which offers ordering services for products and links to other
sites, including IBRIS (International Bryophyte Research Information
Service).
This poster will display up-to-date information on usage of the
service, products available and ongoing work on the bioinformatics
resource provided by the EST sequences.
Visualization of auxin
distribution in Physcomitrella patens
Nicole Bierfreund, Ralf Reski and Eva L. Decker
Plant Biotechnology, Freiburg University, Schaenzlestrasse
1, 79104 Freiburg, Germany
www.plant-biotech.net
The control of auxin distribution is essential for plant development.
In higher plants the auxin flow is well characterized but almost
nothing is known about auxin distribution and its changes during
development in mosses. The presumption ist that auxin builds
up a gradient in the moss protonema. The highest auxin concentration
seems to be in the tip cell and the concentration than gradually
declines to the older cells. In the gametophore nothing is known
about auxin distribution (1, 2).
Our aim is to examine the distribution of auxin in Physcomitrella
patens with a molecular approach. This approach uses an auxin-inducible
reporter gene which represents a molecular marker for monitoring
changes in either auxin concentration or cellular sensitivity
to auxin (3).
For this, Physcomitrella protoplasts were stable transformed
with a construct containing an auxin-inducible gh3 promoter from soybean in front of the ß-glucuronidase gene (gus).
This promoter is rapidly induced only by active auxins.
The auxin distribution is different in various developmental
stages of Physcomitrella. Protonema cells are exhibiting
an auxin gradient from younger to older cells. Buds are expressing
high levels of auxin and leaves are showing auxin accumulation
at the leaf tips and margins.
References
1) Reski, R. (1998). Development, genetics and molecular
biology of mosses. Bot. Acta 111, 1-15.
2) Bopp, M., Atzorn, R. (1992). The morphogenetic system of the
moss protonema, Cryptogam. Bot. 3, 3-10.
3) Li Y., Wu Y. H., Hagen G., Guilfoyle T. (1999) Expression
of the auxin-inducible GH3 promoter/GUS fusion gene as a useful
molecular marker for auxin physiology. Plant Cell Physiol.
40(7), 675-682.
Acknowledgement
Financial support by Deutsche Forschungsgemeinschaft is gratefully
acknowledged.
FURTHER OBSERVATION
OF THE ACTIN NETWORK IN THE MOSS PHYSCOMITRELLA PATENS
Andrija Finka, Didier G. Schaefer and Jean-Pierre Zrÿd
Institute of Ecology, Laboratory of Plant Cell genetics, Biology
Building, UNIL, CH-1015 Lausanne, Switzerland.
According to the general model proposed by Drubin and Nelson
(Drubin and Nelson, 1996), the actin network plays a central
role in the establishment and maintenance of cellular polarity
in eukaryotes. It reinforces the spatial cue perceived by receptors
and interpreted by Rho GTPase and promotes its further propagation
to microtubules and secretory pathways. Therefore, we have decided
to study actin dynamic in our investigations on plant cell polarity
in Physcomitrella.
In plant cells, actin filaments form a net around the nucleus
from which they radiate in bundles to the cell periphery where
they form a branched network of cortical cables. Actin filaments
also co-align with the network of microtubules with one major
exception, the mitotic spindle. The actin network plays an important
role in many polarity related cellular processes. In mitosis,
actin filaments are associated with the microtubule preprophase
band; during cell division they are associated with the growing
cell plate except at the location where cell plate vesicles fuse
to the mother plasma membrane. Involvement of actin filaments
in tip growth is also well documented in root hairs, pollen tubes
and moss filaments: in this case actin filaments are found as
an apical collar like structure which is involved in targeted
transport of vesicles carrying membrane and cell wall material
(for references see (de Ruijter and Emons, 1999)).
The actin network in moss protonema has been studied by rhodamin
phalloidin staining (Doonan and Duckett, 1988). Cortical F-actin
bundles and accumulation of actin at the growing tip of filamentous
cells was detected. The involvement of F-actin in tropic responses
of caulonema cells of Ceratodon purpureus was also shown
using anti-filaments drugs such as cytochalasin-D (Meske and
Hartmann, 1995; Meske et al., 1996). Yet a complete description
of the actin network in moss cells is still missing and this
is probably due to the difficulties to maintain intact actin
structures during cell fixation before staining. The recent development
of a GFP fused to the F-actin binding domain of the mouse talin
protein provides a reliable marker for in vivo studies
of the actin microfilaments (Kost et al., 1998). We have
transformed Physcomitrella with a 35S-GFP-talin cassette and
report here a preliminary in vivo characterisation of
the moss actin network.
In protonemal cells observed through confocal microscopy, GFP
brightly labels a cortical branched network of actin bundles
with an accuracy that was not observed by rhodamin phalloidin
staining. F-actin bundles are aligned parallel to the axis of
the cells and preliminary observation suggests that this network
may be denser in chloronema than in caulonema. Accumulation of
GFP was also detected on both sides of the cell wall separating
2 adjacent filamentous cells as well as at the tip of actively
growing apical cells, confirming thus previous observation suggesting
that actin is actively involved in tip growth. A weaker fluorescence
was also detected that surrounds the nuclei of each cell, indicating
that F-actin forms a net around the nucleus. Finally brightly
labelled cortical star-like structures connected with actin cables
were observed in many cells. These structures correspond to microfilaments
organising centres, which were described in Funaria hygrometrica
(Quader and Schnepf, 1989). Quader and Schnepf have hypothesized
that those actin arrays indicate the position of the next lateral
branch. So far, we have not been able to confirm this hypothesis
and we consider that they may also correspond to the site where
actin polymerisation and cable formation occur.
Freshly isolated GFP-Tn labelled protoplasts do not recover their
spherical form as fast as wild type protoplasts indicating rigidity
of cytoskeleton caused by the binding of GFP-Tn. The expression
of GFP-Tn is drastically diminished during 3-4 initial cell division
following protoplast isolation. A recovery occurs at the 5-6
cells stage of young protonema. In buds, GFP-Tn fluorescence
seems to co-localise with chloroplasts and be absent (or weak)
in the rest of the cell. Developed buds with leaf primordia and
young leaves do not contain any labelled cell. The mature leaves
on the gametophore are uniformly and strongly fluorescent. GFP-Tn
clones are fertile, and spores produced from these plants are
not initially labelled. Protonema and later developmental stages
of moss grown from such spore become to be labelled in the same
manner as those ones derived from protoplasts. The absence of
GFP labelled F-actin, in certain phases of moss development may
indicate that F-actin GFP-Tn complexes are not formed initially
but appear only after the transition periods from unicellular
stage to unidirectional growth and only after the transition
from bud to full caulinary growth.
The use of GFP-talin provides an outstanding tool to visualise
F-actin in vivo in moss cells with an accuracy that cannot
be reached with standard fixation and coloration techniques.
Time lapse video microscopy can describe the dynamic of F-actin
structures during cell growth and cell division. Experiments
with specific inhibitors of microfilaments and microtubules are
conducted to analyse the effect of these drugs on the observed
structures. This work is part of a research program during which
we intend to perform gene disruption experiments of the putative
polarity genes and therefore to be able to directly monitor the
effect of loss of function on the F-actin cytoskeleton.
Acknowledgement
This work is supported by the Swiss National Science Foundation grant # 31-51853.97 to Didier Schaefer and Jean-Pierre Zrÿd
References
de Ruijter, N.C.A. and Emons, A.M.C. (1999) Actin-binding proteins
in plant cells. Plant Biology, 1, 26-35.
Doonan, J.H. and Duckett, J.D. (1988) The bryophyte cytoskeleton:
experimental and immunofluorescence studies of morphogenesis.
, 3, 1-31.
Drubin, D.G. and Nelson, W.J. (1996) Origins of cell polarity.
Cell, 84, 335 - 344.
Kost, B., Spielhofer, P. and Chua, N.-H. (1998) A GFP-mouse talin
fusion protein labels plant actin filaments in vivo and visualises
the actin cytoskeleton in growing pollen tubes. The Plant
Journal, 16, 393-401.
Meske, V. and Hartmann, E. (1995) Reoganization of microfilaments
in protonemal tip cells of the moss Ceratodon purpureus during
the phototropic response. Protoplasma, 188, 59 - 69.
Meske, V., Ruppert, V. and Hartmann, E. (1996) Structural basis
for red light induced repolarisation of tip growth in caulonema
cells of the moss Ceratodon purpureus. Protoplasma, 192,
189 - 198.
Quader, H. and Schnepf, E (1989) Actin filament array during
side branch inititation in protonema cells of the moss Funaria
hygrometrica: an actin organizing center in plasma membrane
Protoplasma, 151, 167 – 170.
Construction of a gametophore-selective
subtraction library in Physcomitrella patens
Sung Hyun Cho, Jeong Sheop Shin
Graduate School of Biotechnology, Korea University, Seoul
136-701, KOREA
The moss Physcomitrella patens is a useful plant to
study plant development and genetics. Since gene knock-out by
homologous recombination is available, it should be focused as
an important plant for functional genomics. The life cycle of
Physcomitrella is composed of gametophyte, the major part
in its whole life, and sporophytic stage. Gametophyte stage is
divided into two stages, the juvenile protonema and the adult
gametophore. Protonemal tissues are branched filaments of single
cells growing by apical extension. Gametophore tissues have stems,
leaves and sex organs, so that it proceeds to sporophyte. Transition
from vegetative to reproductive stage is occurred in gametophore
tissues.
Expressed sequence tag (EST) databases have been established
from many plants, including Arabidopsis thaliana, Zea mays,
Oryza sativa, Brassica napus, etc. Physcomitrella
has also an accumulated EST database over 9,000 EST sequences.
In this study, in order to characterize gametophore-specific
genes and to support nucleotide sequence sources for functional
genomics in Physcomitrella, suppression subtractive hybridization
(SSH), a PCR-based cDNA subtraction technique, was utilized.
SSH reduces false positive ratio because it eliminates any intermediate
step for physical separation of single stranded or double stranded
cDNAs, requires only one subtractive hybridization and achieves
more than 1,000 fold of enrichment for differentially expressed
cDNAs.
Total RNAs were extracted from protonema and gametophore tissue, independently. mRNAs were selected out using magnetic bead (Dynal, USA) and converted to cDNA primed with oligo (dT) primer. A SSH library was constructed with a PCR-select cDNA subtraction kit (Clontech, USA) according to the manufacturerÕs instruction. The constructed SSH library was cloned into TA cloning vectors (Invitrogen, USA). SSH clones were sequenced using an ABI 310 Genetic analyzer (Applied Biosystems, USA) primed with T7, M13 forward or M13 reverse primer according to the thermal cycling protocol of the Taq Dye Terminator Cycle Sequencing kit
(Applied Biosystems, USA). Sequences were compared with GenBank
entries at the amino acid level with the BLASTX subroutine and
at the nucleotide level by the BLASTN subroutine. Matches were
considered significant when the percentage identity was higher
than 40% at the amino acid level or when the BLASTX PAM120 score
was greater than 80.
Among 200 clones, clones that showed high degree of similarities
with other organisms were listed below. Since SSH equalizes whole
genes with low level of expression should be amplified from this
SSH library more than from other normal cDNA libraries. Therefore,
the number of matching sequence with other organism is very low.
Since this SSH library was cloned genes having low degree of
expression as well as gametophore-specific genes, it could contribute
to study for the functional genomics of Physcomitrella.
Visualization of a Cytoskeleton-like
FtsZ Network in Chloroplasts
Justine Kiessling, Sven Kruse, Stefan Rensing, Eva Decker
and Ralf Reski
Plant Biotechnology, Freiburg University, Schaenzlestrasse
1, 79104 Freiburg, Germany.www.plant-biotech.net
It has been a long-standing dogma in cell biology that only
eukaryotic organisms possess a cytoskeleton. This belief was
questioned by the finding that the bacterial cell-division protein
FtsZ resembles tubulin in sequence and structure, and thus may
be the progenitor of this major eukaryotic cytoskeletal element
(1).
Here, we present two nuclear-encoded ftsZ genes of the
moss Physcomitrella patens that are highly conserved in
coding sequence and intron structure. Both their encoded proteins
are imported into plastids and there, like in bacteria, they
act on the division process in a dose-dependent manner.
After transient expression of FtsZ-GFP, we visualized the GFP-tagged
FtsZ1 and FtsZ2 proteins in the chloroplasts of Physcomitrella
protonema cells. While GFP fused to the transit peptide of FtsZ1
is evenly spread within the chloroplasts, FtsZ1-GFP and FtsZ2-GFP
are concentrated to distinct spots. In addition, we identified
filamentous scaffolds inside the chloroplasts that may be involved
in the maintenance of plastid integrity and in plastid division.
The structures differ for FtsZ1 and FtsZ2, thus indicating different
functions for the proteins.
These networks resemble the eukaryotic cytoskeleton and demonstrate
the potential of the tubulin-like FtsZ-family members in forming
different structures within the chloroplasts (2).
As these filaments closely match with REM data on tubulin-like structures in wild-type-plastids from a variety of plants (reviewed in 3), we suggest the term ÒplastoskeletonÓ for these filamentous structures.
 |
 |
Figure 1: Different structures formed by FtsZ1-GFP (left side)
and FtsZ2-GFP (right side).
References
1) Erickson, H. P. (1997). FtsZ, a tubulin homologue in prokaryote
cell division. Trends Cell Biol. 7, 362-367.
2) Kiessling, J., Kruse, S., Rensing, S.A., Harter, K., Decker,
E.L., Reski, R. (2000). Visualization of a cytoskeleton-like
FtsZ network in chloroplasts. J. Cell Biol. 151, 945-950.
3) McFadden, G.I. (2000). Skeletons in the closet. How do
chloroplasts stay in shape? J. Cell Biol. 151,
F19-F21.
Acknowledgement
This work is supported by the Deutsche Forschungsgemeinschaft.
Study on gene flow and
genetic differentiation of populations of marchantialean species
in East Asia using allozyme markers
*Misao Itouga, Tomio Yamaguchi and Deguchi Hironori
Department of Biological science, Graduate school of science,
Hiroshima University,
1-3-1 Kagami-yama, Higashi-hiroshima-shi, Hiroshima-ken 739-8526,
Japan
Spore, gemmae and sperm dispersal restrict gene flow of bryophytes.
Geographic distance of dispersal via spore and gemmae is longer
than that via sperm because movement of sperm is usually limited
by water and consequently spore and gemmae dispersal affect differentiation
of populations. In recent years evolutionary potential of bryophytes
has been measured at the allozyme level. Based on population
genetics, the evolutionary potential is discussed with reference
to genetic diversity within species (Hes) and degree of
genetic differentiation among populations (Gst). Gene
flow is usually estimated using Wright's equation: 4Nm
= (1/Gst) - 1. The Nm value < 1 typically implies
that genetic drift can play a major role in the distribution
of genetic variation among populations. We examined gene flow
and geographic distance for all pairs of populations of marchantialean
species (e.g. Conocephalum japonicum, Reboulia hemisphaerica
ssp. orientalis, Lunularia cruciata) in East Asia
using allozyme markers. Gene flow of C. japonicum populations
was most widespread of all species studied, Nm = 4.188.
Gene flow of Japanese L. cruciata populations was restricted,
Nm = 0.003. Nm value of Reboulia hemisphaerica
ssp. orientalis populations was 1.218. Although maximum
value of geographic distance between C. japonicum populations
was approximately 2700 km, Nm value estimated between
all pairs of C. japonicum populations had nothing to do
with geographic distance. Genetic diversity within C. japonicum
was low, Hes = 0.008 (degree of Hes value of marchantialean
species ranged from 0.008 to 0.167) and genetic differentiation
among C. japonicum populations was seldom found (Gst
= 0.062). Reproductive system of C. japonicum is different
from that of C. conicum (Hes = 0.167) in that the
former tends to produces gemmae and the latter never produce
gemmae. It is highly probable that difference of reproductive
system affects genetic diversity within species. Reproductive
system of Lunularia cruciata is similar to C. japonicum.
Nm value estimated between all pairs of L. cruciata
populations also had nothing to do with geographic distance.
However, Nm value of L. cruciata was extremely
different from that of C. japonicum. This difference is
derived from the degree of genetic differentiation among populations
(Gst). L. cruciata is now considered a naturalize
plant in Japan. We detected five multilocus genotypes (MLG) of
L. cruciata populations in Japan. Gemmae cannot play a
role in long distance dispersal because each population is composed
of only one MLG and spores of L. cruciata populations
in Japan are seldom produced. Geographic distances of Reboulia
hemisphaerica ssp. orientalis populations were long
and gene flow was restricted. Spore dispersal among populations
of R. hemisphaerica ssp. orientalis seems to function
and consequently geographic distance between populations was
> 1000 km, and Nm = 1.218, implying that genetic drift
plays a major role in the distribution of genetic variation among
populations. It is concluded that (1) spore dispersal covers
longer distances than dispersal of gemmae, and (2) Nm
value estimated between all pairs of populations of species with
reproductive system via gemmae had nothing to do with geographic
distance.
Transcriptional Regulation
of Two Chloroplast Superoxide Dismutases, Fe-SOD and CuZn-SOD,
by Copper in a Moss Barbula unguiculata
T. Shiono, T. Miyata, M. Nakata, T. Suzuki, M. Matsuzaki,
I. Yamamoto and T. Satoh
Department of Biological Science, Graduate School of Science,
Hiroshima University, Higashi-Hiroshima, 739-8526, Japan
Bryophytes are considered to have been the most primitive
land plant group and to occupy a critical position for studying
the evolution of oxidative stress adaptation. Then, we have studied
on their isozymes of superoxide dismutases (SODs), which take
a role in reactive oxygen scavenging systems.
There were three types of SOD, Fe-SOD, Mn-SOD and CuZn-SOD with
respect to the prosthetic metal in a moss Barbula unguiculata.
The SOD isozymes of the moss were similar to those of higher
plant and were clearly different from those in the liverwort.
That is, SOD isozyme patterns of the moss and liverwort seemed
to parallel these phylogenetic relationships (Fig. 1).
B. unguiculata had two chloroplast SOD isozymes, Fe-SOD and CuZn-SOD, the expression of which was regulated by copper ion concentration in the culture medium. Fe-SOD activity was expressed in the initial copper concentration less than 0.1µM and repressed more than 1µM. On the other hand, CuZn-SOD activity was expressed more than 1µM. In the liverwort Marchantia paleacea
var. diptera, however, only Fe-SOD was present in the chloroplast and no repression by copper was observed (Fig. 2).Å@
Northern-blot analyses showed the similar results as to the effect
of copper on the expression of the genes for chloroplast Fe-SOD
and CuZn-SOD in the moss. On the other hand, in the liverwort,
the expression of the gene for chloroplast Fe-SOD was not repressed
severly by copper. These results suggested that chloroplast SODs
of the moss and liverwort were regulated by copper ion concentration
at the transcriptional level. The regulation of the gene expression
for chloroplast Fe-SOD in the moss and liverwort might also parallel
these phylogenetic relationships.
Key Words: Superoxide dismutase (SOD), Chloroplast
isozymes, Regulation by copper, Liverwort, Moss
Fig. 1 Phylogenetic distribution and subcellular localization
of three types of SOD in bacteria, algae, bryophytes and plants.
This figure is revised based on the one of Asada (1997) by adding
our information of bryophytes. Cyt, cytoplasmic; Chl, chloroplastic;
Mit, mitchondorial; EC, extracellular.
Fig. 2 Isozymes of SOD in Marchantia paleacea
var. diptera and Barbula unguiculata grown at copper concentrations of 0, 0.1, 1 and 3 µM in media.
Isolation and
characterization of Cab genes from Physcomitrella patens
Seiji Kato, Masakatsu Morita, and Setsuyuki Aoki
Division of Biological Informatics, Graduate School of Human
Informatics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8601, Japan
Physcomitrella patens is expected as a model organism
in plant molecular biology, because efficient transformation
and homologous recombination are applicable to this moss. We
started to study the biological rhythm at the molecular level
in Physcomitrella. No circadian rhythm has so far been
reported in Physcomitrella. We planned to use Cab
(chlorophyll a/b binding protein) genes as markers of circadian
gene expression, because Cab genes have been shown to
exhibit circadian expression widely in plants.
Firstly, we established experimental conditions for non-invasive
measurement of gene expression in Physcomitrella by using
the firefly luciferase gene as a reporter for gene expression.
We fused the luciferase gene downstream of a strong promoter
E7ɶIn (Mitsuhara et al., 1996), and introduced
this fusion into the moss genome. The resulting transformats
showed bioluminescence for more than 48 hours. Thus, the luciferase
gene is expected to be a useful tool for real-time monitoring
of circadian expression of Cab genes in Physcomitrella.
Secondly, we isolated promoters of the Cab genes from
Physcomitrella. The genomic sequence of a Cab gene,
ZLAB1, was already reported in Physcomitrella (Long
et al., 1989). We screened a Physcomitrella genomic
DNA library using a ZLAB1-specific probe. As a result,
we obtained independent five positive clones each of which probably
possesses a distinct member of the moss Cab gene family.
By sequencing genomic DNA inserts of these clones, we found three
different Cab genes (PpCab1, PpCab2 and
PpCab4) including candidate promoter regions. In the upstream
regions of PpCab2 and PpCab4, respectively, we found a motif ÒAAAAATCTÓ, which was reported to be a binding site of CCA1, a putative component of circadian clock in plants. Presently, we are investigating the expression of the Cab
genes using the luciferase reporter gene.
Appearance of
macrochloroplast by ampicillin treatment
in mosses and a fern ally
Hiroyoshi Takano*, Nami Katayama, Motoji Sugiyama, Yoshihiro
Izumi1 and Kanji Ono
Department of Biological Science, Faculty of Science, Kumamoto
University, Kumamoto 860-8555, Japan
1 Faculty of Education and Welfare Science, Oita University,
Oita 870-1192, Japan
* e-mail: takano@kumamoto-u.ac.jp
ß-lactam antibiotics, including ampicillin, form covalent complexes with penicillin-binding proteins (PBPs) in bacteria and, as a result, kill them by interfering with the ability to synthesize cell wall. Only one of these PBPs, PBP3, encoded by the ftsI gene, is essential for prokaryotic cell division (Bramhill, 1997). Recently, it was reported that the treatments of ß-lactam antibiotics resulted in appearance of macrochloroplasts in a moss Physcomitrella patens (Kasten and Reski,
1997). Moreover, ftsI genes were found in chloroplast
genomes of two green algae, Nephroselmis olivacea
and Mesostigma viride (Turmel et al. 1999, Lemieux
et al. 2000). On the other hand, these antibiotics can give no
effect to chloroplast division in higher plant and whole genome
sequence of Arabidopsis thaliana showed that there
was no ftsI homologue.
We studied the effects of ampicillin on the number of chloroplasts per cell in mosses and a fern ally. 100µM ampicillin led to cells with giant chloroplasts in mosses, P. patens and Funaria
hygrometrica. While non-treated cells of P. patens
had about 40-50 chloroplasts, there were less than 10 chloroplasts
in each cell after 4 days of treatment. Fluorescence microscopic
observation of DNA stained with SYBR Green I showed that the
total amount of chloroplast DNA in a treated cell was similar
to that in a non-treated one. Removal of ampicillin at 2 days
caused that cells recovered to have over 40 chloroplasts at 5
days after removal.
Suspension culture of a fern ally, Selaginella nipponica was treated with100µM ampicillin. The growth of S. nipponica
cells was unaffected by the drugs. Without antibiotic, the cells
with one chloroplast were observed in frequencies of 50% after
3 days from the transfer to a fresh medium and then started to
decrease to 1% after 15 days. On the other hand, the rate of
cells with over 3 chloroplasts increased 5% to 70%. The treatment
of ampicillin stopped decreasing of the rate of cells with one
chloroplast and increasing of that of cells with over 3 chloroplast.
These findings suggested that FtsI-homologues have been conserved
in the division apparatus of chloroplasts at the fern ally.
A Novel Gene Family
in Moss (Physcomitrella patens) Shares High Sequence Homology
with TIR-NBS Class of Plant Disease Resistance Genes
Motomu Akita 1, Jari P. T. Valkonen
Department of Plant Biology, Swedish University of Agricultural
Sciences (SLU)
Genetics Centre, PO Box 7080, S-750 07 Uppsala, Sweden
1. Present address: Department of Biotechnological Sciences,
Kinki University
930 Nishimitani, Uchita, Naga, Wakayama 649-6493, Japan
The plant resistance (R) genes are responsible for pathogen
recognition and initiation of signal transduction, which activates
defense genes that condition cells and tissues to inhibit pathogen
invasion. R genes can be divided into several classes based on
different combinations of conserved domains present in the encoded
proteins. The biggest class of R genes encodes for proteins that
have a putative nucleotide binding site (NBS) at the N-proximal
part, and a region (LRR) containing repeated leucine residues
at the C-proximal part. This class can be further divided into
two subgroups based on the motifs present at the N-terminus.
A leucine zipper-like or coiled-coiled motif (LZ/CC) is found
in one subgroup, whereas a domain exhibiting homology with the
Drosophila Toll/mammalian interleukin-1 receptor protein (TIR)
is found in the other subgroup. The aim of this study was to
search for and partially characterize R gene homologues in a
moss (Physcomitrella patens) using the direct PCR-based
cloning approach. We found that this moss contains a family of
R-like genes in which the NBS region shows significant homology
exclusively to the NBS region of plant R genes.
1. Moss has R-like genes that are expressed
The R gene proteins in which several motifs of the NBS are
highly conserved (Fig. 1).
Fig. 1 The typical conserved motifs of the NBS domain in TIR-NBS-LRR
class R genes. The names of motifs are according to Meyers et
al. (1999), including synonymous names (Traut 1994). The motifs
targeted by the degenerate primers used in this study are indicated
in bold.
Using degenerate primers designed according to the NBS domains
of R gene, we found a novel gene family (PpC) that shares high
sequence homology with TIR-NBS class of R gene in P. patens.
At least eight homologous copies of the PpC in the moss genome
were detected and more than four members of this gene family
were found to be expressed. The PpCs contained all of the conserved
motifs that are characteristic with the NBS regions of R genes
(Fig. 1).
Phylogenetic analysis of the deduced amino acid sequences of
the PpCs and R proteins indicated that the PpCs represent a novel
gene family with the closest relationship to the TIR-NBS class
of R genes. No significant similarity to other plant genes than
R genes was observed. These findings may indicate that the TIR-NBS
class of R-like genes evolved prior to diversification of vascular
and non-vascular plants. Our result shed novel light on the evolutionary
history of the R gene families.
2. Expression of PpCs differs at different developmental
stages
Expression level of PpCs was detected by RT-PCR and RNA dot
blotting. The level was significantly different depending on
the developmental stage, as revealed by higher expression in
the gametophyte tissue than in the protonema tissue.
Acknowledgments
We thank Prof. H. Ronne and M. Thelander for interesting discussions
and collaboration on establishing the moss study system in our
laboratory. M. Akita is indebted to S. Vidal, other members of
the SLU Plant Virology Group and colleagues at the SLU Genetics
Centre for materials and help during various parts of the study.
A fellowship and financial support to M. Akita from Kinki University,
Japan, and the grant (SJFR 32.0667/97) to J. Valkonen from the
Forestry and Agricultural Research Council, Sweden, are gratefully
acknowledged.
References
Meyers BC et al. (1999) Plant disease resistance genes encode
members of an ancient and diverse protein family within the nucleotide-binding
superfamily. Plant J 20:317-332
Traut TW (1994) The functions and consensus motifs of nine types
of peptide segments that form different types of nucleotide-binding
sites. Eur J Biochem 222:9-19
The PpMADS1
gene of the moss Physcomitrella patens is expressed in antheridium,
archegonium and developing sporophyte .
Rumiko Kofuji1 and Mitsuyasu Hasebe2
1. Department of Biology, Faculty of Science, Kanazawa University,
Kanazawa 920-1192, Japan, 2. National Institute for Basic Biology,
38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan
MADS-box genes encode a family of transcriptional factors,
some of which play roles as homeotic selector genes for floral
organ development in angiosperms. Recent studies suggest that
the increase in the number of MADS-box genes by gene duplication
and the subsequent recruitment of some MADS-box genes to be expressed
in specific tissues were important for the evolution of complex
reproductive organs. The function of MADS-box genes in gymnosperms
and ferns could not be analyzed, because transformation technique
has not been established. To understand the role of MADS-box
genes in the primitive land plants, three MADS-box genes (PpMADS1,
2 and 3) were cloned from the moss Physcomitrella
patens. PpMADS1 retained the K-box conserved in the
MADS-box genes from seed plants and ferns, but PpMADS2
and 3 had disrupted partial K-box like region. By northern
analyses, PpMADS1 was weakly expressed in haploid protonema
and gametophore. In the moss Physcomitrella patens, homologous
integration is the predominant transformation pathway. We report
here experiment designed to replace PpMADS1 allele with
translational fusions of the coding regions of genomic PpMADS1
to the bacterial uidA gene that encodes b-gulucronidase
(GUS) . In homologous recombinants PpMADS1 promoter activity
was monitored by staining for GUS activity. GUS expression was
shown in gametangia and sporophyte. In developing antheridia
(male gametangia), GUS expression was detected in the formation
of the spermatozoide. In developing archegonia (female gametangia),
expression was detected in the ventral cell and the egg. GUS
expression was detected in not only gametophyte but also sporophyte.
Since the MADS-box genes are expressed in both gametophytes and
sporophytes in moss as well as ferns, recruitment of MADS-box
genes from gametophytic MADS-box genes to sporophytic ones likely
occurred before the ferns evolved after mosses branched from
other land plants.
Structure of chloroplast
genome of the moss Physcomitrella patens
Chika Sugiura1, Yuki Miyata2 and Mamoru Sugita2
1Human Informatics, Nagoya University, Nagoya 464-8601, Japan.
2Center for Gene Research, Nagoya University, Nagoya 464-8602,
Japan.
The structure and gene arrangement of chloroplast genome are
conserved from the moss to the angiosperm, despite their evolutionary
distance and their genome size differences of 121 kb (liverwort)
versus 156 kb (tobacco). Physical and gene map of the Physcomitrella
patens chloroplast DNA was constructed by Calie and Hughes
(1987) and appeared to contain approximately 122 kb organized
into small (21 kb) and large (82 kb) single-copy region separated
by two copies of an inverted sequence (9.4 kb). This overall
structure is almost identical with that of liverwort Marchantia
polymorpha chloroplast (Ohyama et al. 1986). To extend further
our understanding of non-angiosperm chloroplast genomes, we analyzed
the nucleotide sequence of the P. patens chloroplast DNA.
First, we isolated a lambda clone that contained a chloroplast
rDNA operon. Then, we amplified DNA fragments by LA PCR
using the P. patens total cellular DNA and pairs of gene
specific primers. The insert DNA of the lambda clone and the
amplified DNA fragments were shot-gun sequenced. The gene organization
was almost identical to that of the liverwort chloroplast genome
with some gene rearrangements, e.g. relocation of rpoA
from a ribosomal protein gene cluster (rpl23-rpl2 - -
rps11-rpoA). The details of gene organization and RNA
editing of the P. patens chloroplast genome will be presented.
P. patens has been used as a model plant because a high
frequency of transformation and homologous recombination have
been reported. In the present study, we developed chloroplast
transformation in P. patens. Chloroplast trnR-CCG
genes are present in algae and the mosses but not in higher plants,
except black pine. A chimeric aadA gene was inserted into
a trnR-CCG-coding region to make plasmids for chloroplast
transformation. The insert DNAs excised from the plasmids were
introduced in the P. patens protoplasts in the presence
of polyethyleneglycol. Many spectinomycin (spec)-resistant lines
were selected on spec-containing medium. Deletion of trnR-CCG
in the several spec-resistant lines was verified by Genomic Southern
blot analysis and did not affect chloroplast development. This
strongly suggests that trnR-CCG is non-essential under
normal growth conditions.
RNA interference
in Physcomitrella patens
Tomoaki Nishiyama and Mitsuyasu Hasebe
National Institute for Basic Biology, Okazaki 444-8585, Japan
Recent accumulation of Physcomitrella expressed sequence
tags makes a demand for an efficient way to test the function
of each gene.
RNA interferance (RNAi) is specific degradation of mRNA induced
by double strand RNA (dsRNA), first identified in Caenorhabditis
elegans. When dsRNA homologous to a C. elegans gene
is introduced in C. elegans, the mRNA of the gene degrades
and the animal shows a phenotype similar to a loss-of-function
mutant of the gene.
Because introduction of dsRNA in plants cannot be performed as
efficiently as in C. elegans, interferance was induced
in Arabidopsis thaliana by expression of a stem-loop construct
and expression of both sense and anti-sense strands [1]. The
interferance was stronger in the stem-loop construct [1].
With a cDNA fragment at hand, RNAi would be a technology of choice
to analyze the gene function. RNAi will work with relatively
short cDNA sequence, which is advantageous to targeted disruption,
which requires ca. 1 kb of genomic sequence [2]. Additionally,
RNAi can potentially interfere with multiple genes that was duplicated
recently and have a redundant function.
To test if RNAi works in Physcomitrella, a stem-loop construct
of GFP was introduced to a GFP expressing line (GH3-sGFP). As
control, anti-sense and sense constructs were also introduced.
In lines transformed with the stem-loop construct, fluorescence
of GFP was not detected in 11 of 13 lines observed, while 2 lines
continued to express GFP. Fluorescence of GFP was detected in
every line transformed with anti-sense or sense constructs. These
data indicate that RNAi can be efficently induced in Physcomitrella
by expressing stem-loop construct, and the silencing is much
stronger than anti-sense construct, as in Arabdopsis [1].
Acknowledgements
We thank Dr.. Yasuo Niwa for sGFP(S65T), a synthetic gene coding
GFP [3], and Dr. Jon Hughes for the rice actin promotor. The
GH3-sGFP line was kindly provided by Hisako Sakaguchi. We are
grateful to M. Umeda for her technical help.
References
[1] Chiou-Fen Chuang and Elliot M. Meyerowitz 2000
Specific and heritable genetic interference by double-stranded
RNA in Arabidopsis thaliana. PNAS 97: 4985-4990
[2] Didier G. Schaefer 2001
Gene targeting in Physcomitrella patens. Curr. Opin. Plant
Biol. 4:143-150
[3] Wan-ling Chiu, Yasuo Niwa, Weike Zeng, Takanori Hirano, Hirokazu
Kobayashi, and jen Sheen 1996
Engineered GFP as a vital reporter in plants. Curr. Biol. 6:
325-330
Figure. Constructs introduced into a GFP expressing line.
Pactin is a 1.4 kb fragment of rice Actin promoter. PA is a polyadenylation
signal. lacZ is a part of the pUC vector.
Isolation and
characterization of two genes sig1 and sig2 encoding distinct
plastid _ factors from the moss Physcomitrella patens.
Setsuyuki Aoki, Keishi Hara, Masakatsu Morita, Rieko Takahashi,
Mamoru Sugita, and Seiji Kato
Division of Biological Informatics, Graduate School of Human
Informatics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8601, Japan
In higher plants, plastid genes are transcribed by at least
two types of RNA polymerases (RNAPs), a bacteriophage-type single
subunit RNAP and a bacteria-type multisubunit RNAP. The latter
consists of a core catalytic complex and a putative _ factor,
which is likely to confer promoter selectivity on the core complex.
It is supposed that the plastid _ factors regulate different
sets of plastid genes in response to various environmental and
endogenous signals. In spite of the supposed functional significance
as a critical regulator in plastid gene expression, the in
vivo function of plastid _ factors is largely unknown. We
isolated and characterized two genes sig1 and sig2
from the moss Physcomitrella patens, which encode
distinct _ factors PpSIG1 and PpSIG2, respectively. PpSIG1 and
PpSIG2 possess diverged N-terminal halves and C-terminal halves
containing the conserved subdomains 1.2-4.2. Transient expression
assays using a green fluorescent protein showed that the N-terminal
regions of PpSIG1 and PpSIG2 act as chloroplast-targeting signals.
RT-PCR experiments showed that light induces the expression of
the sig1 and sig2 genes. Thus, the PpSIG1 and PpSIG2
are distinct plastid _ factors regulating plastid gene expression
in response to light signals. Phylogenetic analyses indicated
that PpSIG1 and PpSIG2 could be classified into SigB (Cluster
II) and SigA (Cluster I) groups, two major classes of higher-plant
plastid _ factors, respectively.
Reference:
ÒCloning and characterization of the cDNA for a plastid _ factor from the moss Physcomitrella patens.Ó 2001. Keishi Hara, Mamoru Sugita and Setsuyuki Aoki. Biochimica et Biophysica Acta 1517(2):302-306.
Expression Analysis
of FLORICAULA/LEAFY homologue
in the moss, Physcomitrella patens
Takako Tanahashi1, Rumiko Kofuji2, Ryosuke Sano3, Masahiro
Kato1
Mitsuyasu Hasebe3
1Department of Biological Sciences, Graduate School of Science,
The University of Tokyo
2Department of Biology, Faculty of Science, Kanazawa University
3National Institute for Basic Biology
FLORICAULA/LEAFY (FLO/LFY) homologous
genes have been isolated from angiosperms, gymnosperms, ferns,
and fern allies (Himi et al. 2001). Arabidopsis
thaliana LFY positively regulates the expression of some
MADS-box genes that decide floral organ identity. This function
is conserved in gymnosperm FLO/LFY homologues,
while the fern, Ceratopteris richardii FLO/LFY
homologues are not likely to induce MADS-box genes, because the
expression patterns of its FLO/LFY homologues and
MADS-box gene homologues were different. To assess the original
function of FLO/LFY homologues, the moss Physcomitrella
patens was used to characterize the genes using its feasible
gene targeting technique.
We isolated two FLO/LFY homologues in P. patens
(PpLFY1 and PpLFY2) by 3ÕRACE and 5ÕRACE methods using FLO/LFY-specific degenerate primers. PpLFY1
and PpLFY2 were similar to each other (about 90% identical
in coding sequence at the nucleotide level). No other FLO/LFY
homologous genes were detected by genomic southern hybridization
at low stringent condition. Compared with A. thaliana
LFY, the identity were both about 50% (similarity about
78%) at the amino acid level.
We obtained 3 P. patens transgenic lines (L1G-6, 31, 54)
in which uidA (GUS) gene was inserted just before the
PpLFY1 stop codon using homologous recombination. In all
lines, GUS activity was observed in shoot apices, young leaves,
and axillary buds. GUS activity was not detected in protonemata
and rhizoids. This result suggests that PpLFY1 is involved
in the gametophore development at shoot apices. Functional analyses
of PpLFY1 and PpLFY2 by gene disruption are in
progress.
Himi et al. (2001) Journal of Molecular Evolution,
in press.