Comparative genomics of Physcomitrella patens
gametophytic transcriptome and Arabidopsis thaliana:
Implication for land plant evolution
Tomoaki Nishiyama*†, Tomomichi Fujita*†, Tadasu Shin-I‡, Motoaki Seki§¶, Hiroyo Nishide储, Ikuo Uchiyama**,
Asako Kamiya¶, Piero Carninci††, Yoshihide Hayashizaki††, Kazuo Shinozaki§¶, Yuji Kohara‡, and Mitsuyasu Hasebe*‡‡§§*Division of Speciation Mechanisms 2 and 储Computer Laboratory, National Institute for Basic Biology, Okazaki 444-8585, Japan; ‡Genome Biology
Laboratory, Center for Genetic Resource Information, National Institute of Genetics, Mishima 411-8540, Japan; §Laboratory of Plant Molecular
Biology, RIKEN Tsukuba Institute, Tsukuba 305-0074, Japan; ¶Plant Mutation Exploration Team, Plant Functional Genomics Research Group,
RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan; **Research Center for Computational Science, Okazaki National ResearchInstitute, Okazaki 444-8585, Japan; ††Genome Science Laboratory, RIKEN, Wako 352-0198, Japan; and ‡‡Department of Molecular
Biomechanics, SOKENDAI, Okazaki 444-8585, JapanThe mosses and flowering plants diverged >400 million years ago.
The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land
plants have been inferred to be haploid-dominant, suggesting thatgenes used in the diploid body of flowering plants were recruitedfrom the genes used in the haploid body of the ancestors during
the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella
patens, and compared the moss transcriptome to the genome of
Arabidopsis thaliana. We constructed full-length enriched cDNA
libraries from auxin-treated, cytokinin-treated, and untreatedgametophytes of P. patens, and sequenced both ends of >40,000
clones. These data, together with the mRNA sequences in the
public databases, were assembled into 15,883 putative transcripts.
Sequence comparisons of A. thaliana and P. patens showed that atleast 66% of the A. thaliana genes had homologues in P. patens.
Comparison of the P. patens putative transcripts with all known
proteins, revealed 9,907 putative transcripts with high levels ofsimilarity to vascular plant genes, and 850 putative transcripts withhigh levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana
genome, supporting the evolutionary hypothesis. Our study also
revealed that a number of genes are moss specific and were lost in
the flowering plant lineage.

Green plants first expanded their habitat to the land by theearly Silurian (430 million years ago), and subsequently
diverged into various lineages with different morphologies (1).
The bryophytes and the ancestor of vascular plants diverged
early in land plant evolution. Mosses are bryophytes, and theirmorphologies and life cycles differ significantly from those offlowering plants (2, 3). The sporophyte (diploid) generation isdominant over the gametophyte (haploid) generation in flowering plants, whereas the opposite occurs in mosses. Leafy shoots
differentiate in the diploid generation of flowering plants, andthe gametophytes, i.e., pollen tubes and embryo sacs, are epiphytic to the diploid plant bodies, in which fertilization occurs.
Mosses propagate by means of spores, from which protonemata,which are filamentous cells, germinate, grow, and differentiate
gametophores with haploid leafy shoots. The tissues of thegametophytic leafy shoots of mosses are much simpler than the
sporophytic shoots of flowering plants. Archegonia and antheridia differentiate at the tips of the leafy shoots to form eggs
and sperm, respectively. Moss sporophytes are epiphytic to thehaploid leafy shoots.
Despite the divergent morphologies and life cycles of flowering plants and mosses, the molecular mechanisms of certain
physiological processes, such as the light- and abscisic acid
(ABA)-signal transduction networks, are preserved. The phytochrome gene, which encodes the red兾far-red light receptor andwww.pnas.org兾cgi兾doi兾10.1073兾pnas.0932694100

is well characterized in flowering plants, has been cloned frommosses (4, 5). Phototropic responses, which are implicated in thelight-associated signal transduction network, have also beenreported in mosses (6). The desiccation stress response network
mediated by ABA is probably also conserved between mosses
and flowering plants (7). Furthermore, auxin and cytokinin areimportant developmental regulators in both flowering plants
and mosses (reviewed in refs. 8 and 9), though their respectiveroles in development differ in these two plant forms because ofdevelopmental and morphological differences.
The differences and similarities between flowering plants andmosses should be reflected in their genomes. Almost the entire
genome sequence of Arabidopsis thaliana has been determined(10). Therefore, comparisons of the genes found in A. thaliana
and mosses should provide evolutionary insights into the numberof shared and lineage-specific genes among these different
lineages.
Physcomitrella patens is a widely used model because it has thehighest reported ratio of homologous recombination to nonhomologous recombination of all land plants (11). A number of P.
patens genes have been isolated and disrupted successfully(12–16). In addition to these individual gene analyses, EST
projects are ongoing, and P. patens is the most suitable moss forgenetic comparisons with the flowering plants. The first P. patens
EST library, which contained 82 ESTs, was reported by Reski etal. (17), and this was followed by a second library of 169 ESTs
(18). The Physcomitrella EST Programme (www.moss.leeds.
ac.uk兾) is an ongoing project, and 18,000 ESTs have been
deposited to date in GenBank (www.ncbi.nlm.nih.gov兾dbEST兾
dbEST㛭summary.html). Ralph Reski’s group has accumulated⬎100,000 ESTs (ref. 19, www.plant-biotech.net兾Rensing㛭et㛭al㛭transcriptome2002.pdf), though these data are not deposited inpublic databases. Additional EST data are required for detailed
analysis of the similarities and differences between the A. thaliana
and P. patens genomes.
In this study, we constructed full-length enriched cDNA
libraries of P. patens from auxin- and cytokinin-treated gametophytes, as well as from gametophytes that were grown without
exogenous plant hormones. These phytohormones act at various
stages of P. patens development, and libraries of clones that havebeen treated with these hormones should cover a broad range oftranscriptome. We combined the 85,191 new ESTs that wedetermined with other publicly available P. patens ESTs, andorganized them into 15,883 putative transcripts. These seAbbreviations: MST, moss transcripts absent in vascular plants; NAA, naphthalene acetic
acid; BA, 6-benzylaminopurine.
†T.N. and T.F. contributed equally to this work.
§§To whom correspondence should be addressed. E-mail: mhasebe@nibb.ac.jp.

PNAS 兩 June 24, 2003 兩 vol. 100 兩 no. 13 兩 8007– 8012

PLANT BIOLOGY

Communicated by Peter R. Crane, Royal Botanic Gardens, Kew, Surrey, United Kingdom, May 6, 2003 (received for review December 26, 2002)

quences were compared with those of A. thaliana and otherorganisms. Based on the results of these studies of genetic
content, we discuss the evolution of land plants.

Table 1. Base composition of each position in the 5ⴕ end
sequences

Materials and Methods

1
2
3
4
10
20

Plant Materials, RNA Preparation, and Construction of Full-Length
cDNA Libraries. P. patens (Hedw.) Bruch and Schimp subspecies

patens Tan (20), collected in Gransden Wood, Huntingdonshire,
U.K. (21), was used as the wild-type strain. The protonemata
were ground with the Polytron (Kinematica, Littau, Switzerland), and inoculated into BCDATG medium (22), BCD medium (22) that contained 1.0 mM CaCl2 and 1.0 ␮M naphthalene
acetic acid (NAA; Sigma), or BCD medium that contained 1.0
mM CaCl2 and 0.50 ␮M 6-benzylaminopurine (BA; Sigma) for
the untreated, NAA-treated, and BA-treated specimens, respectively, at 25°C under continuous light, and the tissues were
harvested at 13–14, 8–11, and 8–13 days, respectively. At harvest,
the untreated tissues contained chloronemata and young gametophores with two to five leaves, the NAA-treated tissues
contained chloronemata, caulonemata, and rhizoid-like protonemata, and the BA-treated tissues contained chloronemata,caulonemata, and malformed buds.
Total RNA was extracted from each tissue sample as described(23), and poly(A)⫹ RNA was purified with oligo(dT) magnetic
beads (DynaBeads; Dynal A.S., Oslo). The cDNA was synthesized by using trehalose-thermoactivated reverse transcriptase(24), and full-length cDNA was recovered by using the biotinylated CAP trapper method (25, 26). The ␭-full-length cDNA
vector (27) and the single-strand linker ligation method (28)were used in the construction of the cDNA libraries. One roundof normalization (29) was performed to construct the NAA- andBA-treated cDNA libraries to reduce the frequency of highly
expressed mRNAs in the libraries.
DNA Sequencing. In the preliminary analyses, 908 clones werechosen from the untreated library, and the DNA was sequenced
from the 5⬘-end. For mass sequence analyses, the clones from theuntreated, NAA-treated, and BA-treated libraries were arrayedin 35, 50, and 48 384-well-plates, respectively, and the clones
were sequenced at both ends. Low-quality sequences were
removed based on PHRED quality score (30) so that average scoreis at least 20. The sequence data from six plates were inconsistentin terms of their 5⬘ and 3⬘ end sequences because of mishandling;
therefore, the reverse sequences were renamed and treated as
independent sequences during later analyses.
Sequence Data Sets. In addition to the 85,191 ESTs sequenced inthis study, all of the P. patens mRNA entries in GenBank release131.0 (excluding our ESTs) were extracted. ESTs with theannotation of ‘‘High quality sequence stop’’ were trimmed to thelength specified by the annotation. All of the A. thaliana protein
sequences were retrieved from the RefSeq database (31) andused in the A. thaliana protein data set. The Synechocystis sp.
PCC 6803, Saccharomyces cerevisiae, and Drosophila melanogaster coding sequences and translated sequence data sets wereobtained from the following locations.
ftp:兾兾ftp.kazusa.or.jp兾pub兾cyanobase兾Synechocystis兾Synecho.
p.nt.gz
Y ftp:兾兾ftp.kazusa.or.jp兾pub兾cyanobase兾Synechocystis兾Synecho.
p.aa.gz
Y ftp:兾兾genome-ftp.stanford.edu兾pub兾yeast兾yeast㛭ORFs兾orf㛭
coding.fasta.Z
Y ftp:兾兾genome-ftp.stanford.edu兾yeast兾yeast㛭protein兾yeast㛭nrpep.
fasta.Z
Y ftp:兾兾ftp.fruitfly.org兾pub兾genomic兾fasta兾na㛭gadflyCDS.dros.
RELEASE2.Z
Y

8008 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0932694100

Position

A

G

C

T

%purineT兾C

13,425
5,752
6,194
6,646
7,276
7,769

21,682
12,47011,992
12,396
11,342
11,058

4,836
7,865
8,826
6,667
10,996
11,3253,164
17,006
16,10017,39013,44812,939

81
42
42
44
43
44

0.65
2.16
1.82
2.61
1.22
1.14

The occurrence of each base was counted at each position. The %purine
was calculated as 100 ⫻ (A ⫹ G)兾(A ⫹ G ⫹ C ⫹ T).

Y

ftp:兾兾ftp.fruitf ly.org兾pub兾genomic兾fasta兾aa㛭gadf ly.dros.
RELEASE2.Z

The nonredundant protein sequence data set was obtained fromftp:兾兾ftp.ncbi.nih.gov兾blast兾db兾nr.Z.
Identification of Taxonomic Positions. The taxonomic classificationof the organisms followed that of the National Center for
Biotechnology Information taxonomy database (32). The Geninfo Identifier (GI) number for the taxonomy ID (tax ID) table
was obtained from ftp:兾兾ftp.ncbi.nih.gov兾pub兾taxonomy兾
gi㛭taxid㛭prot.dmp.gz, and the tree structure was according to‘‘nodes.dmp’’ in ftp:兾兾ftp.ncbi.nih.gov兾pub兾taxonomy兾taxdump.tar.gz. The tree was traversed from a node that was
identified by the tax ID to the root until a specified node wasreached. When the root was reached, the taxonomic position was
unidentified and thus investigated manually.

Statistical Test of Transcript Representation in Different Libraries.

The number of clones in each library was counted for eachputative transcript. We used the null hypothesis that a putativetranscript is equally represented in the three libraries andtherefore the proportion of clones belonging to the putativetranscript is constant in the three libraries. We used the G testof independence (33).
Results and Discussion
Library Quality. Full-length cDNA clones are important, not only
for gene annotation and the determination of transcriptional
start sites, but also for functional analyses (34, 35). Recently,
methods have been developed that allow preferential cloning of
cDNA that corresponds to full-length mRNAs with 5⬘-endproximal cap structures (reviewed in ref. 36). These methods
have been used in large-scale analyses of transcripts from human(37), mouse (38, 39), fruit fly (40), rice (39), and A. thaliana(41, 42).
We constructed three full-length enriched cDNA libraries
from the gametophytic tissues of P. patens that were NAAtreated, BA-treated, or untreated, and we selected 19,200,
18,432, and 14,348 clones, respectively, from the libraries. The
clones were sequenced from both ends. In total, 4.78 ⫻ 107
nucleotides of 85,191 sequences remained after the eliminationof low-quality sequences and contaminated clones showing
nearly complete match to Escherichia coli or Dictyostelium
discoideum sequence.
We analyzed the proportion of full-length cDNA clones that
contained mRNA start sites. Because transcription usually starts
at a purine base (43), we examined the base composition of thefirst and subsequent nucleotides at the 5⬘ end of each 5⬘
sequence. The first nucleotide showed a strong bias for purine(81% G or A; Table 1), whereas 42–44% of the 2nd to 20th
nucleotides were purines. If the cDNAs were not full-length anddid not contain the first nucleotide of the mRNA, then the basecomposition of the first nucleotide would have been identical to
Nishiyama et al.

Assembling the EST Data into Contigs and Estimating the Gene
Number. P. patens mRNA sequence data from other sources, suchas the ESTs from the Physcomitrella EST Programme, wereobtained from GenBank (release 131.0) and pooled with ourdata. After removing low-quality sequences, which were identified by their annotations, and vector and linker sequences, whichwere excluded based on sequence similarity searches, a data setof 5.4 ⫻ 107 total nucleotides in 102,553 sequences was obtained.
The data were assembled with the CAP3 Sequence Assembly
program (46). We obtained 22,885 contigs in total. Of these,
20,589 were represented in our library, whereas the remaining
2,296 contigs arose from other sources. Because our ESTs wereobtained by sequencing each clone from the 5⬘- and 3⬘-ends, we
can assign 5⬘- and 3⬘-contigs to the original clone. In total, 814
of the 20,589 contigs had ⬎5% ambiguity (N), and were represented by only one sequence (singlet). These contigs were notused for pairing, because they may not represent independenttranscripts. By using the data on the 5⬘- and 3⬘-ends, theremaining 19,775 contigs were organized into 13,593 putativetranscripts. We assigned a unique identifier (of the form
Pnnnnnn, where n is a digit) to each of the 13,593 putativetranscripts. Each of the 2,296 contigs from other sources, withthe exception of six contaminating sequences from bacteriophage ␭, was regarded as an independent putative transcript. The
addition of these 2,290 putative transcripts to the 13,593 putativetranscripts reveals that at least 15,883 putative transcripts are
expressed in P. patens.
Although ⬎100,000 sequences were determined for the 22,885
contigs, 9,417 of the 22,885 contigs were composed of only one
sequence, i.e., either an EST or a deposited mRNA sequence.
This finding indicates that the EST library is not saturated, andthe sequencing of more clones will probably reveal new genes. In
addition, gametophytes with gametangia and sporophytes were
not sampled. Therefore, the total number of P. patens transcripts
probably exceeds 15,883.
Nishiyama et al.

Some Genes Are Represented Differently in the Three Libraries.

Because we sequenced three libraries, we were able to identify
genes that were represented differently in each library. We used
the G test (33) to assess whether putative transcripts were overor underrepresented in the auxin- and cytokinin-treated libraries. Putative transcripts of this type contained genes that wereregulated by auxin and cytokinin, as well as genes that wererelated to differentiation and development induced by the auxin
and cytokinin treatments.
Seven PpIAA1 cDNA (14) clones were found in the auxintreated library and one clone was found in the cytokinin-treatedlibrary. In this case, the null hypothesis of equal representationin the three libraries was rejected at the 1% level. Induction of
the PpIAA1 gene by auxin is consistent with the reported RNA
gel blot analysis (14).
Similarly, all of the putative transcripts were tested for overor under-representation in each library. Sixty and sixty-eightputative transcripts were over-represented at the 0.1% level in
the auxin- and cytokinin-treated libraries, respectively (Tables 5
and 6, which are published as supporting information on the
PNAS web site). One and twenty-one putative transcripts were
under-represented in the auxin- and cytokinin-treated libraries,
respectively (Table 7, which is published as supporting information on the PNAS web site).
Of the putative transcripts that were overrepresented in thecytokinin-treated library, two (P013130 and P006794) showed
high-level similarity to cytokinin oxidase, which functions incytokinin degradation (47). The promoter of the cytokinin
oxidase gene DSCKX1 of Dendrobium cv. Sonia was shown to becytokinin-inducible (48). Overrepresentation of the cytokinin
oxidase gene in the cytokinin-treated library is consistent withinduction by cytokinin, and suggests that mosses and vascularplants share common mechanisms for the regulation of cytokinin
levels. The receptor-like kinases (P007265, P008576, P001848,
and P006061) and the ethylene responsive element binding
protein (EREBP)-like transcription factors (P012262 andP001581) were also overrepresented in the cytokinin-treatedlibrary, though the functions of these factors are unknown.
Many of the A. thaliana Genes Have Homologues in the Moss
Gametophytic Transcriptome. To analyze the proportion of A.

thaliana genes that were expressed in the moss gametophytes, all26,178 amino acid sequences encoded by A. thaliana genes wereobtained from the RefSeq database (31) at the National Center
for Biotechnology Information and used as the query sequences
in a similarity search with TBLASTN (45) to the moss data set. The
peak occurred at 0.1 ⬍ E ⱕ 10 and the tail appeared at E ⱕ 1e-3
(Fig. 2, which is published as supporting information on the
PNAS web site). If we regard the P. patens contigs with E ⱕ 1e-3
as homologues of A. thaliana query sequences, 17,382 of 26,178(66.4%) of the A. thaliana genes have homologues in P. patens.
Random matches with an E value ⱕ 1e-3 would be expected tooccur only 26.2 (26,178 ⫻ 0.001) times. Matches occurred at amuch higher frequency than this, and most were considered tobe caused by homology.
TBLASTN similarity searches were also performed by using theamino acid sequences of the 26,178 A. thaliana genes as query
sequences and the sequence data sets from budding yeast (S.
cerevisiae), fruit fly (D. melanogaster), and cyanobacterium (Synechocystis sp. PCC 6803); putative homologues (E ⱕ 1e-3) werediscovered at frequencies of 40.5%, 47.1%, and 21.3%, respectively. The overlaps of A. thaliana genes with homologues indifferent organisms are summarized in the Venn diagram in Fig.
1. Of the A. thaliana genes, 5,039 showed significant similarities
to P. patens genes, but not to genes of the other three organisms.
Sixteen classes of plant-specific transcription factors have beenreported in the A. thaliana genome (10). Of these, the MYB
R2R3 (49), homeodomain-leucine zipper (50), and ARF-AUX兾PNAS 兩 June 24, 2003 兩 vol. 100 兩 no. 13 兩 8009

PLANT BIOLOGY

that of nucleotides in subsequent positions. Although terminaltransferase activity of the reverse transcriptase, adding C to the3⬘ end of cDNA (44), may have contributed partly to overrepresentation of G, this does not account for the increase of A.
Therefore, the deviation of first nucleotide base composition
from that of nucleotides at subsequent positions suggests that ahigh proportion of the cDNA clones are full-length. In our 5⬘ end
sequence data (Table 1), the T兾C ratio at the first position (0.65)
differed from that at the second (2.2) and subsequent (1.1–2.6)
positions. This finding implies that a significant proportion of the5⬘-end-proximal pyrimidine bases also represent mRNA startsites.
To estimate the proportion of clones that contained complete
coding sequences, we examined two P. patens tubulin-␣ genes (Y.
Sato, T.F., and M.H., unpublished data) and all of the 38 P.
patens mRNA deposited in GenBank (release 130.0) that wereannotated as containing complete coding sequences. ESTs corresponding to the genes were identified with BLASTN (45)searches and then tested whether they had 5⬘ or 3⬘ UTR. The
search revealed that 31 genes had corresponding clones in our
EST library (Table 4, which is published as supporting information on the PNAS web site, www.pnas.org). In the 5⬘ end
sequences, 95.4% (456 of 478) of the clones contained 5⬘ UTR,
and 97.8% (480 of 491) of the 3⬘ end sequences contained 3⬘
UTR (Table 4). If we assume that 5⬘ and 3⬘ end truncation
occurred independently, the proportion of clones that had both
start and stop codons can be calculated as the product of the
proportion of clones complete at the 5⬘ and 3⬘ ends. Therefore,
we estimated that 93% (95.4% ⫻ 97.8%) of the clones containedcomplete coding sequences.

Table 2. Taxonomic distribution of best hits
Taxonomic position of the best hit
Vascular plants
Nonvascular land plants
Green algae
Metazoa
Fungi
Other eukaryotes
Archaea
Cyanobacteria
Other bacteria
Viruses
Synthetic construct

No. of putative transcripts
9,907
58
51
311
60
48
12
91
214
2
3

The putative transcripts of P. patens were subjected to a BLASTX searchagainst the nonredundant dataset, and the organism (excluding the mosses)that carried the best-hit gene was determined and sorted according to taxonomic position.

Fig. 1. Classification of A. thaliana genes according to the presence ofhomologous genes in other organisms. The amino acid sequences of all of
the predicted genes of A. thaliana were used as the query in a TBLASTN
similarity search with the moss, yeast, fruit fly, and cyanobacterial data sets.
The outermost circle represents all of the A. thaliana genes. The inner
circles, which are labeled Moss, Eukaryotes, and Cyanobacterium, represent genes that have similarity to moss contigs, budding yeast and fruit fly
coding sequences, and cyanobacterium coding sequences, respectively.
The areas depicted are not proportional to the actual gene numbers,
and the number of A. thaliana genes in each category is written in eachsegment.

IAA (14) genes have been reported, and these genes were also
found in the moss EST data. The NAC-like, zinc finger-DOF,
zinc finger-WRKY, AP2兾EREBP, ABI3兾VP1-like and GRASlike transcription factors were found in the EST data set. The
putative transcript P011588 showed high-level similarity to
Mybst1, which is a Myb single-repeat protein, which suggests that
P011588 also encodes a Myb single-repeat protein, though thecomplete sequence is required to verify that P011588 encodes
only a single Myb domain. The remaining six classes of transcription factors (cycloidea-like, YABBY, EIN3, RAV-like,squamosa-binding protein, and GT1-like) were not identified in
the P. patens EST data set.
Some gene families, such as the REL-homology region protein, nuclear steroid receptors, forkhead-winged helix, and POU
were found in metazoans, but were absent from the A. thaliana
genome (10) and P. patens EST data set.
Because 17,382 (66.4%) of the predicted A. thaliana genes had
related sequences in the P. patens ESTs and the ESTs are notsaturated, the number of genes with P. patens homologues should
be higher. This observation indicates that the P. patens gametophytic transcriptome is substantially similar to the A. thaliana
genome. Although the A. thaliana genome sequence has been
largely determined, the functions of many of the predicted genes
are still unknown. A number of A. thaliana genes have homologues in P. patens, but not in the yeast or fruit fly. Because genetargeting is feasible in P. patens, functional analyses of thehomologues in P. patens should increase our understanding ofgene function in flowering plants.
P. patens Expresses Genes That Are Not Present in the A. thaliana
Genome. We assessed whether the closest relatives of moss genes

existed in vascular plants or in other organisms. The putative
moss transcripts were used as the query sequence in a BLASTX(45) search against the A. thaliana protein data set and thenonredundant amino acid sequence (nr) data set in GenBank. In
8010 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0932694100

total, 9,942 (62.6%) of the 15,883 putative transcripts had
significant similarities (E ⱕ 1e-3) to A. thaliana proteins, and10,757 (67.7%) had significant similarities to the nr data set. Thisis substantially higher than the estimation by Rensing et al. (19)that only half of the P. patens genes have known homologue inplants, fungi, animals, or bacteria, despite the fact that we used
only gametophytic transcripts. This discrepancy is perhaps because we connected 5⬘ and 3⬘ contigs into a single putativetranscript, which was used for the search. When we searched withthe contigs as query sequences, half of the contigs showed nosignificant similarity to known proteins, which is consistent to theresult by Rensing et al. (19).
We sorted the 10,757 putative transcripts into groups based on
the organisms that had the highest levels of similarity to the mosstranscripts. Of these, 9,907 transcripts had highest levels ofsimilarity to genes in vascular plants, whereas 850 showed highest
levels of similarity to genes in other organisms (Table 2 andTables 8 and 9, which are published as supporting informationon the PNAS web site). Some of the 850 putative transcripts
showed similarities to both vascular plants and other organisms,with low levels of difference. Because the significance of thesedifferences could not be ascertained, we selected 300 of 850
putative transcripts with E values that were at least 1e7-foldlower than the E values for best hits with vascular plants. These
are candidates for transcripts that are absent in vascular plants
but present in mosses. We termed these 300 putative transcripts
MSTs (moss transcripts absent in vascular plants). Thirteenof the 300 MSTs were found in liverworts, which are also
bryophytes.
The 300 MSTs probably correspond to new genes in land
plants. Genome sequencing projects with several vascular plants
are underway. Therefore, genes with high levels of similarity tosome of the 300 MSTs may be found in future genomic analyses.
Nonetheless, we believe that the 300 MSTs may be attributed
primarily to gene loss or rapid evolution in vascular plantlineages, or in some cases, to horizontal gene transfer to P. patensfrom other organisms. Sequence conservation among the mosses
and other organisms implies that the genes are important inmosses, whereas genes with lower or null levels of similarity in
the A. thaliana genome either have changed function or are notrequired in flowering plants, which likely include genes that are
involved in moss-specific processes.
Characterization of Moss Transcripts That Are Not Present in Vascular
Plants. The 9,907 putative transcripts with the highest levels ofsimilarity to vascular plants corresponded to 6,773 genes in thenonredundant data set, and the 300 MSTs corresponded to 244
genes. The gene ontology (GO) (51) categories of biological

Nishiyama et al.

Common to Absent in
vascularvascularplantsplantsNo. of putative transcripts
No. of corresponding genes in the nr data set
No. of genes with GO assigned
Metabolism, %Transport, %Signal transduction, %Cell organization and biogenesis, %Cell cycle, %Photosynthesis, %Response to external stimulus, %Response to stress, %Cell growth and兾or maintenance, %Cell death, %Others, %

9,907
6,773
2,946
77.7
13.2
2.7
1.3
1.1
1.0
0.8
0.5
0.4
0.4
0.8

300
244
116
68.0
19.8
4.7
1.7
0.9
0.9
1.1
0.3
1.7
0.0
0.9

nr, nonredundant.

processes were assigned to 2,946 of the 6,773 genes and 116 of
the 244 genes, respectively, based on INTERPRO (52) entries thatmatched the amino acid sequences of the 6,773 and 244 genes.
The remaining 3,827 and 128 genes did not match INTERPRO
entries. The frequencies of the GO categories at the fourth levelare summarized in Table 3. The composition of the annotations
was generally similar between the 2,946 and 116 genes, in that the‘‘metabolism’’ category appeared with the highest frequency; thepercentage of genes in this category was lower in the 116-genegroup (68.0%) than in the 2,946-gene group (77.7%). The
category ‘‘metabolism’’ was divided into subcategories; of thesethe ‘‘protein metabolism’’ and ‘‘biosynthesis’’ subcategories had
fewer representatives among the 116-gene group (12% and 7%of metabolism, respectively) than among the 2,946-gene group(20% and 14%, respectively; Table 10, which is published as
supporting information on the PNAS web site). These results
suggest that genes that are involved in protein metabolism andbiosynthesis are well conserved between mosses and vascularplants. By contrast, the group of 116 genes had a higher
proportion of genes that were associated with transport andsignal transduction than the group of 2,946 genes (Table 3). Thisfinding may be related to the fact that most cells of mosses,
especially the protonemata, are in direct contact with the outsideenvironment.
Examples of Moss Transcripts That Are Absent in Vascular Plants. P.
patens is known for its extraordinarily high ratio of homologous
recombination to nonhomologous recombination. Some of thegenes that had the highest levels of similarity to genes fromorganisms other than vascular plants were related to DNA
damage repair, i.e., the P011933 gene for the DNA repairhydrolase, the P002718 gene for RecA, and the P009868 gene for
Eso1. P009868 showed high-level similarity to the Eso1 gene of
the fission yeast Schizosaccharomyces pombe, with a score of 120
bits and an E value of 7e-27. Eso1 is essential for sister chromatid
cohesion (53), which is important for the efficient homologous
recombinational repair of DNA double-strand breaks, because
the intact sister chromatid paired to the damaged chromatid
serves as a repair template (54, 55). Eso1 is a fusion of DNApolymerase ␩, which functions in DNA damage repair, and thesister chromatid cohesion protein (53). Although a high level ofsimilarity was found within the DNA polymerase ␩ domain, it
remains to be seen whether the moss gene has the sister
chromatid cohesion domain, because we obtained only the end
sequences. DNA damage repair may be more important during
Nishiyama et al.

the haploid stage than during the diploid stage, because only one
copy of each gene exists in the haploid, whereas an extra copy
exists in the diploid cell. Analysis of these genes may unveil thereason why P. patens has such a high rate of homologous
recombination.
The putative transcripts P001256 and P005876 were highly
similar to the chlorophyll a兾b-binding protein (cab) homolog
LI818r-1 of Chlamydomonas reinhardtii. The cab homologue
LI818 was first identified in Chlamydomonas eugametos as alight-inducible mRNA that encoded a new class of the cab genefamily (56). This class of cab homologues has not been reported
in other land plants, and may have been lost during the evolutionof the vascular plant lineage.
P006275 is an example of a gene that was probably transferredfrom bacteria, because it showed highest similarity to the glutatione S-transferase of Mesorhizobium loti (NP㛭105706), with ascore of 251 bits and an E value of 9e-66. P006275 also had
similarities with a number of glutathione S-transferases fromother bacteria. The eukaryotic gene with highest similarity to
P006275 was the glutatione S-transferase gene of Emericellanidulans, with 158 bits and 1e-37, whereas the closest gene ingreen plants was the glutatione S-transferase gene of Glycinemax, with 29 bits and 1e-10. Clones of P006275 were found in all
three of our libraries, and also in two libraries from the Physcomitrella EST Programme project, which indicates that thissequence is not a contaminant. The absence of a closely related
homologue in plants was further tested in a TBLASTN search thatmatched the amino acid sequence of P006275 with the EST data
set. The best non-P. patens hit was an EST from Porphyra
yezoensis, which is a red alga, with 108 bits and an E value of
2e-22; no ESTs from green plant species were found, despite theexistence of extensive EST libraries from several seed plants.
Phylogenetic analysis (Fig. 3, which is published as supporting
information on the PNAS web site) also supports a bacterial
origin for P006275.
Genes of Sporophyte- and Gametophyte-Dominant Plants. Based on
the phylogenetic analyses, the closest relatives of the land plants
are the freshwater green algae of Charophyceae (57). The single
diploid cells of charophycean algae are zygotes, which undergo
meiosis to produce four haploid gametophytic cells. Within theland plant lineage, bryophytes diverged at the most basal position. Both charophycean algae and bryophytes have gametophyte-dominant life cycles, which suggests that the ancestors ofland plants, from which sporophyte-dominant vascular plants
evolved, had gametophyte-dominant life cycles. Because themulticellular sporophytes probably evolved during land plant
history from the gametophyte-dominant plants in the absence ofmulticellular diploid plant bodies, it seems reasonable to speculate that the genes that function in sporophytes were recruitedfrom genes that functioned in the gametophytes of the common
ancestor of mosses and flowering plants. However, there is no
direct evidence for this evolutionary scenario, because gene
expression in gametophytes of land plants is not well characterized, even in model plants such as A. thaliana, in which almost
the whole genome has been sequenced. The general similarity
between P. patens gametophyte ESTs and the A. thaliana genome
supports the evolutionary scheme that proposes the recruitmentof gametophytic genes into the sporophyte generation during
land plant history, though we do not have an accurate estimationof the number of A. thaliana genes that are expressed insporophyte generation. Analysis of the genes that are expressedin A. thaliana gametophytes will provide further insights into theevolution of the gametophyte and sporophyte generations.

Conclusions
The gametophyte transcriptome of P. patens is similar to theA. thaliana genome. Our findings that ⬎66% of A. thaliana
PNAS 兩 June 24, 2003 兩 vol. 100 兩 no. 13 兩 8011

PLANT BIOLOGY

Table 3. Gene Ontology (GO) assignment to putative transcripts

genes have homologues in P. patens gametophytes, and that
⬎90% of the most closely related homologues of P. patens
gametophytic transcripts occur in vascular plants, suggest that
gemetophytes and sporophytes use similar gene sets. On theother hand, 850 putative transcripts of P. patens showed lower
levels of similarity to genes of A. thaliana and vascular plants
than to genes in other organisms, thereby indicating that thesegenes are probably associated with moss-specific features, suchas morphology, life cycle, responses to the environment,
metabolism, and other aspects. Analyses of these genes will
give further evolutionary insights and new genetic resources
for agricultural purposes.
The functions of many A. thaliana genes have not yet been
revealed. Because gene targeting is feasible in P. patens, analysisof related genes in P. patens will be a good way of achieving an
understanding of the function of plant-specific genes. Our
full-length cDNA collection is a valuable resource for functional
genomics in plants and is distributed by the RIKEN Bio Resource

Center (www.brc.riken.go.jp兾lab兾epd兾Eng. The data can be accessed through internet at http:兾兾moss.nibb.ac.jp兾.

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8012 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0932694100

We thank K. Oishi, S. Haga, T. Morishita, S. Miura, F. Ohta, H. Hayashi,
S. Nishizaka, H. Nomoto, and M. Sano for EST sequencing. We thankDrs. A. Kitayama and N. Ueno for providing equipment and valuable
discussions. We also thank M. Naruse, Y. Bitoh, and Y. Dodo fortechnical assistance. This work was mainly supported by a Grant-in-Aid
for Scientific Research on Priority Areas (C) ‘‘Genome Science’’ from
the Ministry of Education, Culture, Sports, Science and Technologyof Japan (MEXT) (to M.H., Y.K., M.S., and K.S.), Grant-in Aid for
Scientific Research on Priority Areas (Grants 14036229 and 13044001)
from MEXT (to M.H. and T.F.), a Grant-in-Aid from MEXT (to M.H.
and T.F.), and Japan Society for the Promotion of Science (JSPS) Grant
RFTF00L0162 (to M.H. and T.F.). Construction of the full-length library
was also supported by a grant for Genome Research from RIKEN, theProgram for Promotion of Basic Research Activities for Innovative
Biosciences, the Special Coordination Fund of the Science and Technology Agency, and a Grant-in-Aid from MEXT (to K.S.). T.N. is a JSPS
Fellow.

Nishiyama et al.