Annual Report 2002
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DIVISION OF SPECIATION MECHANISMS II

Professor:HASEBE, Mitsuyasu
Associate Professor:MURATA, Takashi
Assistant Professor:FUJITA, Tomomichi
Technical Staff:SUMIKAWA, Naomi
NIBB Research Fellow:MISHIMA, Misako (-March 31)
HIWATASHI, Yuji
JSPS Research Fellow:NISHIYAMA Tomoaki
SATOH, Yoshikatsu
Graduate Students:KOBAYASHI-ARAKAWA
Satoko
SAKAKIBARA, Keiko
SAKAGUCHI, Hisako
(Shinshu Univ.)(-March 31)
NAKAMURA, Tohru
(Niigata Univ.)(April 1-)
Technical Assistant:TANIKAWA, Yukiko
BITOH, Yoshimi
NARUSE, Mayumi
AOKI, Etsuko
WATANABE, Kyoko (-Feb. 4)
YANO, Kana (-Nov. 8)
KABETANI, Keiko (Feb. 1-)
OGURA, Youko (Nov. 1-)
Secretary:KABEYA, Kazuko
Visiting Scientists:Jean-Pierre Zrÿd 1)(-Jan. 18)
1)from Université de Lausanne, Lausanne, Switzerland

All living organisms evolved from a common ancestor more than 3.5 billion years ago, and accumulated mutations on their genomes, which caused the present biodiversity. The traces of evolutionary processes are found in the genomes of extant organisms. By comparing the genomes of different organisms, we can infer (1) the phylogenetic relationships of extant organisms and (2) the genetic changes having caused the evolution of morphology and development. The inferred phylogenetic relationships give important insights on problems in various fields of evolutionary biology, and our group focuses on biogeography, evolution of morphological traits, and systematics in wide range of taxa. On the evolution of morphology and development, we aim to explore genetic changes led the evolution of plant body plan. We selected Arabidopsis (angiosperm), Gnetum (gymnosperm), Ginkgo (gymnosperm), Ceratopteris (pteridophyte), Physcomitrella (bryophyte), and some green algae as models to compare the gene functions involved in development of both reproductive and vegetative organs in land plants.

I. Molecular phylogeny of the sundews Drosera

Carnivorous plants have long attracted the attention of botanists, because of their highly specialized morphology and curious trapping mechanisms. However, their evolutionary processes are still unknown. The sundew genus Drosera consists of carnivorous plants with active flypaper traps and contains nearly 150 species mainly distributed in Australia, Africa, and South America, with some species in the Northern Hemisphere. In addition to the confusion of intrageneric classification of Drosera, intergeneric relationships among Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda are problematic. We conducted phylogenetic analyses of DNA sequences of the chloroplast gene rbcL for 59 species of Drosera covering all sections except one. These analyses revealed that 5 sections in 11 including 3 monotypic sections are polyphyletic. Combined data of the rbcL and 18S rDNA sequences were used to infer phylogenetic relationships among Drosera, Dionaea, and Aldrovanda. This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, suggesting that snap traps of Aldrovanda and Dionaea are homologous despite their morphological differences. MacClade reconstructions indicated that multiple episodes of aneuploidy occurred in a clade including mainly Australian species, although chromosome numbers in other clades are not so variable. D. regia native to South Africa and most species native to Australia were basally clustered, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that Australian species expanded their distribution to South America, and then to Africa. Expansion of distribution to the North Hemisphere from the South Hemispere occurred in a few different lineages.

Figure.1. The most parsimonious tree resulting from parsimony analysis of the combined sequences of rbcL and 18S rDNA. Branch lengths correspond to the number of nucleotide substitutions (ACCTRAN optimization). Numbers above branches represent bootstrap values more than 50% of 10,000 bootstrap replicates, and numbers below branches are decay indecies .(Bremer,1988).

II. Evolution of reproductive organs in land plants

A flower is the most complex reproductive organ in land plants and composed of sepals, petals, stamens, and gynoecium. Female haploid reproductive cells are covered with a sporangium (nucellus) and two integuments, and further enclosed in a gynoecium. Male haploid reproductive cells (pollens) are covered with a sporangium (pollen sack). On the other hand, gymnosperms and ferns have simpler reproductive organs than angiosperms and lack sepals and petals. Female sporangia (nucellus) of gymnosperms are covered with only one integument. Sporangia of ferns have no in-teguments and are naked on the abaxial side of a leaf.

The development of floral organs is mainly regulated by A-, B-, C-function genes, which are members of the MIKC-type MADS-box genes. These genes are transcription factors containing four domains, MADS, I, K, and C domains. MADS-box genes of angiosperms are divided into more than 10 groups based on the gene tree. The FLO/LEAFY gene is the positive regulator of the MADS-box genes in flower primordia.

What kind of changes of the MADS-box genes caused the evolution of the complex reproductive organs in the flowering plant lineage? Comparisons of MADS-box and FLO/LFY genes in vascular plants suggest that the following sequential changes occurred in the evolution of reproductive organs. (1) Origin of MIKC-type MADS-box genes. (2) The number of MADS-box genes increased, and the three ancestral MADS-box genes that later generate A-, B-, C-functions genes were likely originated before the divergence of ferns and seed plants. (3) Specification of MADS-box gene expression in reproductive organs occurred in seed plant lineage. (4) The ancestral gene of the AG group of MADS-box genes acquired the C-function before the divergence of extant gymnosperms and angiosperms. (5) The gene duplication that formed the AP3 and PI groups in MADS-box genes occurred before the diversification of extant gymnosperms and angiosperms. (6) The ancestral gene of angiosperm A-function gene was lost in extant gymnosperm lineage. (7) LFY gene became positively regulate MADS-box genes in the common ancestor of angiosperms and gymnosperms after the divergence of ferns and seed plants. (8) Spatial and temporal patterns of A-, B-, C-function gene expression were established in the angiosperm lineage.

Figure 2. Expression of Physcomitrella patens MADS-box gene. GUS activity of PpMADS1-uidA protein was detected in transgenic Physcomitrella patens. Localization of PpMADS1-GUS protein in an archegonium (a-d), and an antheridium (e-h) is shown.

MIKC-type MADS-box genes have been reported only from vascular plants, but not from non-vascular plants. To investigate the origin of MIKC-type MADS-box genes, eight MADS-box genes (PPM1-PPM5 and PPMADS1-3) were characterized in the moss Physcomitrella patens (Henschel et al. 2002). Phylogenetic reconstructions and comparison of exon-intron structures revealed that these moss genes represent two different classes of homologous, yet distinct MIKC-type MADS-box genes. We named them MIKCc-type and MIKC*-type genes. MIKCc-type genes are abundantly present in all land plants, and include the A-, B-, and C-function genes. In contrast, LAMB1 from the clubmoss was identified as the only other MIKC*-type gene published so far. Our findings strongly suggest that the last common ancestor of mosses and vascular plants contained at least one MIKCc- and one MIKC*-type gene.

III-I. Evolution of vegetative organs

The ancestor of land plants was primarily haploid. The only diploid cell was the zygote, which immediately underwent meiosis. It is believed that early during land plant evolution, zygotic meiosis was delayed and a multicellular diploid sporophytic generation be-came interpolated into the life cycle. In the early stages of land plant evolution, sporophytes are epiphytic to gametophytes, as observed in extant bryophytes. During the course of evolution, both generations started to grow independently at the stage of pteridophytes. Finally gametophytes became much reduced and epiphytic to sporophytes in seed plant lineage. Molecular mechanisms of development in a diploid generation have been well studied in some model angiosperms, but we have scarce information on those in a gametophyte generation. For example, mosses have leaf- and stem-like organs in their haploid generation, but it is completely unknown whether similar genes involved in angiosperm leaf and stem development are used in the gametophytic generation of mosses or not. To understand the evolution of body plans in diploid and haploid generation at the molecular level, we focus on the comparison of molecular mechanisms governing shoot development between Arabidopsis and the moss Physcomitrella patens. Physcomitrella is known by its high rate of homologous recombination and suitable for analyze gene functions using the gene targeting, and should be a good model lower land plants.

III-II. Characterization of homeobox genes in the moss Physcomitrella patens

Homeobox genes encode transcription factors involved in many aspects of developmental processes including shoot development in angiosperms. The homeodomain-leucine zipper (HD-Zip) genes, which are characterized by the presence of both a homeodomain and a leucine zipper motif, form a clade within the homeobox superfamily and previously re-ported only from vascular plants. We isolated 10 HD-Zip genes from P. patens (Pphb1-10 genes). Based on a phylogenetic analysis of the 10 Pphb genes and previ-ously reported vascular plant HD-Zip genes, all the Pphb genes except Pphb3 belong to three of the four HD-Zip subfamilies (HD-Zip I, II, and III), indicating that these subfamilies originated before the divergence of the vascular plant and moss lineages. Pphb3 is sister to HD-Zip II subfamily, and has some distinctive characteristics, including the difference of a1 and d1 sites of its leucine zipper motif, which are well conserved in each HD-Zip subfamily. Comparison of the genetic divergence of representative HD-Zip I and II genes showed that the evolutionary rate of HD-Zip I genes was faster than HD-Zip II genes.

The moss homologs of SHOOTMERISTEMLESS and ZWILLE genes, which are involved in Arabidopsis shoot development, have been cloned and their characterization is in progress.

III-III. Establishment of enhancer and gene trap lines in the moss Physcomitrella patens

We also established enhancer and gene trap lines and tagged mutant libraries of P. patens to clone genes involved in the leafy shoot development in haploid generation. Elements for gene-trap and enhancer-trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter, respectively. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene-trap and 1073 enhancer-trap lines were obtained from 5637 and 3726 transgenic lines, respectively. Expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene-trap line YH209, which shows rhizoid-specific expression, was obtained by 5’ and 3’ RACE. This gene was named PpGLU, and forms a clade with plant acidic alpha-glucosidase genes. Thus, these gene-trap and enhancer-trap systems should prove useful to identify tissue- and cell-specific genes in Physcomitrella.

Publication List:

Henschel, K., Kofuji, R., Hasebe, M., Saedler, H., Munster, T. and Theissen, G. (2002) Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens. Mol. Biol. Evol. 19, 801-814.

Imaizumi, T., Kadota, A., Hasebe, M. and Wada, M. (2002) Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens. Plant Cell 14, 373-386.

Ishikawa, H., Ueno, Y., Semiarti, E., Onouchi, H., Kojima, S., Tsukaya, H., Hasebe, M., Soma, T., Ikezaki, M., Machida, C. and Machida, Y. (2002) The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel family of proteins characterized by cysteine repeats and a leucine zipper. Plant Cell Phys. 43, 467-478.

Kitakura, S., Fujita, T., Ueno, Y., Terakura, S., Wabiko, H. and Machida, Y. (2002) The protein encoded by oncogene 6b from Agrobacterium tumefaciens interacts with a putative transcription factor in tobacco cells.Plant Cel14, 451-463.

Maggio, A., Miyazaki, S., Veronese, P., Fujita, T., Ibeas, J.I., Damsz, B., Narasimhan, M.L., Hasegawa, P.M., Joly, R.J. and Bressan R.A. (2002) Does proline accumulation play an active role in stress-induced growth reduction? Plant J. 31, 699-712.

Murata, T., Karahara, I., Kozuka, T., Giddings Jr., T.H., Staehelin, L.A. and Mineyuki, Y. (2002) Improved methodd for visualizing coated pits, microfilaments, and microtubules in cryofixed and freeze-substituted plant cells. J. Electron microscopy 51, 133-136.

Rivadavia, F., Kondo, K., Kato, M. and Hasebe, M. (2003) Phylogeny of the sundews, Drosera (Droseraceae) based on chloroplast rbcL and nuclear 18S ribosomal DNA sequences. Amer. J. Bot. 90, 123-130.


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