Isolation of Homeodomain–Leucine Zipper Genes from the Moss Physcomitrella patens and the Evolution of Homeodomain–Leucine Zipper Genes in Land Plants

Keiko Sakakibara, Tomoaki Nishiyama, Masahiro Kato and Mitsuyasu Hasebe

*National Institute for Basic Biology, Okazaki, Japan;
{dagger}Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki, Japan; and
{ddagger}Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Homeobox genes encode transcription factors involved in many aspects of developmental processes. 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 were previously reported only from vascular plants. Here we report the isolation of 10 HD-Zip genes (named Pphb1Pphb10) from the moss Physcomitrella patens. Based on a phylogenetic analysis of the 10 Pphb genes and previously reported vascular plant HD-Zip genes, all of 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 the HD-Zip II subfamily and has some distinctive characteristics, including the difference of the 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 that of HD-Zip II genes.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Homeobox genes are transcription factors characterized by the homeodomain (McGinnis et al. 1984Citation ; Scott and Weiner 1984Citation ), which usually consists of 60 conserved amino acid residues that form the helix-loop- helix-turn-helix structure that binds DNA (reviewed in Laughon 1991Citation ). These genes play crucial and diverse roles in many aspects of development, including the early development of animal embryos, the specification of cell types in yeast, and the initiation and maintenance of the shoot apical meristem in flowering plants (reviewed in Bürglin 1998Citation ). The homeobox genes are structurally divided into two groups: the typical homeobox genes and the TALE homeobox genes. The TALE homeobox genes have three more residues between helix 1 and helix 2 than the typical homeobox genes. Members of both groups have been reported from metazoa, fungi, and green plants, and the homeobox genes likely split into the two groups in the common ancestor of these three groups of multicellular organisms (Bharathan et al. 1997Citation ; Bürglin 1997, 1998Citation ).

Many homeobox genes have been isolated from vascular plants. They are classified into seven groups based on their amino acid sequence similarities: KNOX, BELL, ZM-HOX, HAT1, HAT2, ATHB8, and GL2 (Bharathan et al. 1997Citation ). The KNOX and BELL genes belong to the TALE homeobox genes, while the other genes belong to the typical homeobox genes (Bharathan et al. 1997Citation ; Chan et al. 1998Citation ). The HAT1, HAT2, ATHB8, and GL2 genes are characterized by the leucine zipper motif (Landschulz, Johnson, and McKnight 1988Citation ) adjacent to the C-terminus of the homeodomain and have been renamed the HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV subfamilies, respectively (Meijer et al. 1997Citation ; Aso et al. 1999Citation ).

The functions of the HD-Zip genes are diverse among the different subfamilies, and even within the same subfamily (Chan et al. 1998Citation ). HD-Zip I and II genes are likely involved in signal transduction networks of light (Carabelli et al. 1993, 1996Citation ; Schena, Lloyd, and Davis 1993Citation ; Aoyama et al. 1995Citation ; Steindler et al. 1997, 1999Citation ), dehydration-induced ABA (Söderman, Mattsson, and Engström 1996Citation ; Frank et al. 1998Citation ; Lee and Chun 1998Citation ; Söderman et al. 1999Citation ), or auxin (Kawahara, Komamine, and Fukuda 1995Citation ). These signal transduction networks are related to the general growth regulation of plants. The overexpression of sense or antisense HD-Zip I or II mRNA usually alters growth rate and development (Schena, Lloyd, and Davis 1993Citation ; Aoyama et al. 1995Citation ; Meijer et al. 1997Citation ). Most members of the HD-Zip III subfamily play roles in cell differentiation in the stele (Baima et al. 1995Citation ; Zhong and Ye 1999Citation ), although the functions of some genes remain unknown (Sessa et al. 1998Citation ; Aso et al. 1999Citation ). HD-Zip IV genes are related to the differentiation of the outermost cell layer (Rerie, Feldmann, and Marks 1994Citation ; Cristina et al. 1996Citation ; Lu et al. 1996Citation ; Masucci and Schiefelbein 1996Citation ; Ingram et al. 1999Citation ; Kubo et al. 1999Citation ).

HD-Zip genes have been reported only from vascular plants (Bharathan et al. 1997Citation ; Aso et al. 1999Citation ) and are not found in the entire genome of either the metazoan Caenorhabditis elegans or the fungus Saccharomyces cerevisiae. Since genes with either a leucine zipper motif or a homeodomain have been reported in metazoa, fungi, and green plants, Schena and Davis (1992)Citation speculated that the HD-Zip genes originated in the green plant lineage by exon shuffling between a gene encoding a homeodomain and another gene encoding a leucine zipper motif. Although there have been a large number of studies of angiosperm HD-Zip genes, little is known about these genes in other green plants, and the origin of the HD-Zip genes is not yet well understood. Aso et al. (1999)Citation reported HD-Zip I, II, and III genes from the fern Ceratopteris richardii and indicated that the four HD-Zip subfamilies had already diverged before the divergence between the angiosperm and fern lineages, approximately 400 MYA (Stewart and Rothwell 1993Citation , p. 510). In addition to these studies, analyses of HD-Zip genes in other land plants and green algae are necessary to further understand the evolution of the HD-Zip gene family.

Physcomitrella patens is a moss. Mosses diverged from the vascular plant lineage approximately 430 MYA (Stewart and Rothwell 1993Citation , p. 510). Unlike vascular plants, the haploid gametophyte generation is dominant in mosses. Dominance of the haploid generation is also conspicuous in the charophyceaen algae, the green algae most closely related to the land plants (Graham and Wilcox 2000Citation , p. 499). The body plans and life histories of mosses and vascular plants differ extensively. For example, unlike vascular plants, mosses form simple leaf- and stem-like organs in their gametophyte generation (Hébant 1977Citation ; Bold, Alexopoulos, and Delevoryas 1987Citation , pp. 270–276, 314–334; Reski 1998Citation ). We chose to study P. patens because it is well developed as a model organism (Cove, Knight, and Lamparter 1997Citation ; Reski 1998Citation ), and this will enable further studies on gene function.

In this study, we isolated 10 HD-Zip genes from P. patens and included these in a phylogenetic analysis with previously reported HD-Zip genes isolated from vascular plants. We used the gene tree to determine the phylogenetic relationships of the HD-Zip genes and whether four HD-Zip subfamilies were present in the last common ancestor of the mosses and vascular plants. The difference in evolutionary rates between the HD- Zip I and II subfamilies is reported, and the reason is discussed.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Strains and Culture Conditions
Physcomitrella patens (Hedw.) Bruch & Schimp subsp. patens Tan collected in Gransden Wood, Huntingdonshire, U.K. (Ashton and Cove 1977Citation ), was used in this study as the wild-type strain. Physcomitrella patens was grown on medium based on BCD medium (D. J. Cove, personal communication), which contains 1 mM MgSO4, 10 mM KNO3, 45 µM FeSO4, 1.8 mM KH2PO4 (adjusted pH to 6.5 with 4 M KOH), and trace elements (Knight et al. 1988Citation ). The trace elements included 0.22 µM CuSO4, 0.19 µM ZnSO4, 10 µM H3BO4, 0.10 µM Na2MoO4, 2 µM MnCl2, 0.23 µM CoCl2, and 0.17 µM KI. Growth supplements were added to the BCD medium for spore germination (10 mM CaCl2 and 5 mM diammonium (+)–tartrate; S medium), for protonemata culture (1 mM CaCl2 and 5 mM diammonium (+)–tartrate; P medium), and for induction and culture of gametophores (1 mM CaCl2; G medium). These media were solidified with 0.8% (w/v) agar (A- 9799, SIGMA, St. Louis, Mo.) in 9-cm petri dishes. The solidified medium was covered with a layer of cellophane (Futamura Chemical Industries Co., Ltd., Nagoya, Japan) to facilitate collection of the moss from the medium (Grimsley, Ashton, and Cove 1977Citation ). Sporangia, each containing a few thousand spores, were stored at 4°C for several weeks to several years. The sporangia were sterilized with 1% (w/v) sodium hypochlorite (Wako, Osaka, Japan) for 5 min and rinsed four times with sterilized water. The sterilized sporangia were squashed with a sterile stick in a 1.5-ml plastic tube containing 1 ml of sterilized water, and 200 µl of this spore suspension was sown on the S medium. The dishes were stored at 25°C under continuous light (40 µmol photons/m2/s). The spores usually germinated 2 or 3 days after inoculation, and protonemata developed. The protonemata were collected 2 weeks after germination and were ground with a Polytron homogenizer (Kinematica, Littau, Switzerland) in approximately 8 ml of sterilized water at a speed setting of 4, and 2 ml of the suspended protonemata were transferred onto the P medium. To induce gametophores, the suspended protonemata were transferred to the G medium and grown at 25°C under continuous light (40 µmol photons/m2/s) for 1 month after inoculation.

Cloning the HD-Zip Genes
Using ISOGEN (Nippon Gene, Tokyo, Japan), total RNA was extracted from the wild-type protonemata 1 week after inoculation and from gametophores grown for 1 month after inoculation of protonemata onto G medium. Complementary DNA was synthesized from the total RNA using the 3' RACE system (Life Technologies, Inc., Rockville, Md.). PCR amplifications were performed according to Aso et al. (1999)Citation using a homeodomain-specific primer, HB1 (Bürglin et al. 1989Citation ) or HD1 (Aso et al. 1999Citation ). The PCR products were separated on 1% (w/v) agarose gels (SeaKem GTG, FMC BioProducts, Rockland, Maine). The DNA fragments between 0.5 and 2 kb were purified with the GeneClean III kit (BIO 101 Inc., La Jolla, Calif.), and cloned using the CLONEAMP pAMP1 system (Life Technologies, Inc.). In all, 326 clones were obtained using HB1 (96 from the protonema cDNA and 230 from the gametophore cDNA), and 84 clones were obtained using HD1 (30 from the protonema cDNA and 54 from the gametophore cDNA). These candidate clones were sorted into 58 groups according to their digestion patterns using AluI, and then two or three clones from each group were sequenced with the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, Calif.) or the Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer Applied Biosystems) using an ABI PRISM 377 DNA sequencer (Perkin-Elmer Applied Biosystems). The 5' region of each gene encoding a homeodomain was cloned using the 5' RACE system (Life Technologies, Inc.) and sequenced. The nucleotide sequences of the gene-specific primers are deposited in the DNA database, with the sequence of each gene as additional information. Total genomic DNA was extracted from the wild-type protonemata 1 week after inoculation using PHYTOPURE (Amersham Pharmacia Biotech, Uppsala, Sweden) and further purified using the CTAB method (Murray and Thompson 1980Citation ).

Two gene-specific primers located close to each end of the putative mRNA were synthesized. Nested PCR amplifications were performed with the cDNA or genomic DNA using these primers to obtain cDNA and genomic DNA clones. The PCR product was cloned into pBluescriptII SK+ (Stratagene, La Jolla, Calif.) or pGEM3z (Promega, Madison, Wis.). To exclude PCR errors, at least two clones obtained from independent PCR were sequenced for each gene. When discrepancies were found, the majority from at least three independently amplified PCR products was selected.

Phylogenetic Analysis
Plant HD-Zip genes similar to P. patens HD-Zip genes were obtained from the nr and dbest data sets at NCBI using the programs blastx or tblastn (version 2.0.10) (Altschul et al. 1997Citation ). Genes containing undetermined amino acid residues within the HD-Zip domain were not used for further analysis. Nucleotide sequences were translated to amino acid sequences based on the universal code. Genes with identical amino acid sequences in the region used for the analysis were treated as a single operational taxonomic unit. The deduced amino acid sequences of 96 HD-Zip genes used in this study were aligned using CLUSTAL W, version 1.8 (Thompson, Higgins, and Gibson 1994Citation ), and then revised manually. Eighty-four amino acid sites without indels were used for the phylogenetic analysis.

To search for the maximum-likelihood (ML) tree, we used neighbor-joining (NJ) and most-parsimonious (MP) trees as start trees for a local rearrangement search (Adachi and Hasegawa 1996Citation ). NJdist and ProtML programs were in the MOLPHY, version 2.3b3, package (Adachi and Hasegawa 1996Citation ). An NJ tree (Saitou and Nei 1987Citation ) was obtained with NJdist based on the ML distance under the JTT model (Jones, Taylor, and Thornton 1992Citation ) calculated with ProtML. MP trees were obtained by heuristic searches with tree bisection-reconnection (TBR) using PAUP*, version 4.0 b4a (Swofford 2000). Three kinds of heuristic search were performed: (1) a heuristic search from a simple addition sequence without the steepest-descent option, (2) 84 searches from a random tree without the steepest-descent option, and (3) 10 searches from a random tree with the steepest-descent option. These analyses found 205,787 trees with 1,330 steps. The likelihoods of all these trees were calculated using ProtML under the JTT model, and the trees were sorted according to their Akaike information criterion (AIC) values (Adachi and Hasegawa 1996Citation ). The best 1,000 trees were subjected to further local rearrangement searches (Adachi and Hasegawa 1996Citation ). Further phylogenetic analyses constraining a monophyletic relationship of HD-Zip I genes were performed. The approximate likelihood (Adachi and Hasegawa 1996Citation ) of all of the 6,081,075 trees obtained under this constraint was calculated. For the best 10,000 trees, log likelihood was calculated. An ML tree found in this search was further subjected to a local rearrangement search.

The local bootstrap probability of each branch was estimated by using the resampling-of-estimated-log-likelihood (RELL) method (Kishino, Miyata, and Hasegawa 1990Citation ; Hasegawa and Kishino 1994Citation ).

Difference in Evolutionary Rates Between the HD-Zip I and the HD-Zip II Subfamilies
We examined whether the evolutionary rate in the HD-Zip I subfamily was higher than that of the HD-Zip II subfamily. Rate is the distance divided by the divergence time. If we focus on ortholog pairs of two defined species, the divergence time is a constant, and the rate can be compared by comparing the distance. Every pair of species that has ortholog pairs in both the HD-Zip I and the HD-Zip II subfamilies was used. The ortholog pairs were determined according to tree topology and species phylogeny. For each pair (i), the evolutionary distance (di) was calculated with ProtML under a JTT model using the -D option. The distance (di, dj) of two pairs (i, j), one from the HD-Zip I subfamily and the other from the HD-Zip II subfamily, were compared. To test whether the distance was different between pairs i and j, i.e., di > dj, the difference di - dj (Dij) was tested against 0. Dij was calculated for each set of amino acid data resampled in a bootstrap test, and standard deviations were calculated from 10,000 bootstrap samples.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Isolation of HD-Zip Genes from P. patens
Candidate HD-Zip cDNA clones obtained by the 3' RACE system using the homeobox-specific primers were assigned to 10 genes, named Pphb1Pphb10 (P. patens homeobox genes; the accession numbers for the DNA database are AB028072–AB028080 and AB032182, respectively). The 5' regions of the Pphb genes were obtained using the 5' RACE system with gene-specific primers for each HD-Zip gene. Every Pphb gene encodes both a homeodomain and a leucine zipper motif. The latter motif is adjacent to the C-terminus of the homeodomain (fig. 1 ), and one of four amino acid residues (leucine, isoleucine, valine, or alanine) usually appears every seven amino acids in the motif ("d" positions in fig. 2 ). One or two acidic regions, expected to be the transcriptional activation domains (Ptashne 1988Citation ), were found adjacent to the N- terminus or C-terminus of the homeodomain and leucine zipper motif in every Pphb gene except Pphb10 (fig. 1 ). Putative repression domains, which were reported from Oshox1 as high-alanine/proline/glutamine-content regions (Meijer et al. 1997Citation ), were not found in any Pphb genes. The CPSCE motif and C-terminal end consensus reported in HD-Zip II genes (Chan et al. 1998Citation ) were found in Pphb4 (fig. 1 ). The genomic regions of Pphb3, Pphb4, Pphb5, Pphb7, and Pphb8 were amplified with primers located at the 5' or 3' end of each gene. Based on the comparison of the cDNA and genomic regions of these Pphb genes, the locations of introns were inferred and are shown in figure 1 .



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Fig. 1.—The structure of Pphb1Pphb10 cDNAs. The circle and "A (n)" indicate the positions of the translation termination codon and the poly (A)+ tail, respectively. Genomic regions corresponding to the cDNA of Pphb3Pphb5, Pphb7, and Pphb8 were sequenced, and positions of introns are indicated by triangles. White, black, gray, hatched, and crosshatched boxes indicate the homeodomain, leucine zipper motif, acidic region, CPSCE motif, and C-terminal end motif, respectively. Pphb10 is separated into two lines connected by a slash

 


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Fig. 2.—Alignment of amino acid sequences of the HD-Zip genes used in this study. Dashes indicate gaps. The names of the subfamilies are shown to the left. Pphb genes cloned from Physcomitrella patens are shown in bold type. The letters ag indicate the position of each residue within the heptad of the leucine zipper motif, and typical amino acid residues (leucine, isoleucine, valine, or alanine) for the leucine zipper motif are usually observed in "d" positions. Asterisks above the alignment indicate the 85 amino acids used for the phylogenetic analysis. White and black boxes above the alignment indicate the homeodomain and leucine zipper motif, respectively. The black lines below the white box indicate helices 1, 2, and 3, respectively. Accession numbers of genes are as follows: Athb9 (Y10922), Athb14 (Y11122), Ifl1 (AF188994), Crhb1 (AB013791), Pphb10 (AB032182), Athb8 (Z50851), ATML1 (U37589), Phahox (U34743), AHDP (U85254), ANL2 (AAD47139), OCL1 (CAB51059), Hahrs1 (L76588), Glabra2 (L32873), CHB2 (D26574), CHB4 (D26576), Hahb1 (L22847), Athb13 (AAF20996), Athb3 (X62644), HAT7 (U09340), Athb6 (X67034), CHB6 (D26578), Athb5 (X67033), Hdl56 (AAF01764), Oshox4 (AAD37697), Athb7 (X67032), Athb12 (AF001949), Oshox6 (AAD37699), HBLZP (AAD38144), Pphb5 (AB028076), Pphb6 (AB028077), Pphb2 (AB028073), Pphb1 (AB028072), Pphb9 (AB028080), Pphb8 (AB028079), Pphb7 (AB028078), Vahox1 (X94947), CHB3 (D26575), Athb1 (X58821), HAT5 (M90416), Oshox5 (AAD37698), CHB1 (D26573), Hdl57 (AAF01765), Crhb6 (AB013796), Crhb11 (AB013801), Crhb8 (AB013798), Crhb4 (AB013794), Crhb5 (AB013795), Crhb7 (AB013797), Athb4 (Y09582), HAT3 (U09338), HAT1 (U09332), HAT2 (U09335), Gmh1 (X92489), PHZ1 (X95193), PHZ2 (X94375), PHZ4 (X94449), Athb2 (X68146), HAT4 (M90394), Oshox1 (X96681), THOM1 (S76820), Oshox7 (AAD37700), Oshox2 (AAD37695), HAT14 (U09334), Crhb10 (AB013800), Crhb3 (AB013793), Crhb2 (AB013792), Crhb9 (AB013799), HAT9 (U09342), HAT22 (U09336), Hahb10 (L48485), Cphb2 (AJ005833), Pphb4 (AB028075), Cphb1 (AJ005820), Oshox3 (AAD37696), Pphb3 (AB028074)

 
Phylogenetic Analysis of the HD-Zip Genes
Eighty-six vascular plant HD-Zip genes obtained from the DNA database and the 10 Pphb genes were aligned (fig. 2 ), and 84 amino acid residues indicated in figure 2 were used for phylogenetic analysis (EMBL accession number ds42823). Of the 84 sites, 73 sites were informative in parsimony analyses. We searched the ML tree using the NJ and MP trees as start trees for local rearrangements. The ML trees obtained from the NJ and MP trees had log likelihoods of -7,167.98 and -7,160.21, respectively. In the ML tree obtained from the MP trees, the HD-Zip I subfamily unexpectedly does not form a monophyletic group, while the HD-Zip I subfamily is always monophyletic, as in previous studies (e.g., Chan et al. 1998Citation ; Aso et al. 1999Citation ). Finally, a tree with a log likelihood of -7,155.93 ± 512.44 and an AIC of 14,689.55 was obtained. The ML tree contains some short branches, and its AIC becomes better (14,673.63) when these branches are collapsed. The ML tree with collapsed branches is shown in figure 3 .



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Fig. 3.—The maximum-likelihood tree of the 96 HD-Zip genes found by local rearrangement search. Local bootstrap probability is shown on or below branches where available. This is an unrooted tree. The horizontal branch length is proportional to the estimated evolutionary distance. The species from which the gene was isolated is indicated by an abbreviation after the gene name: Ath (Arabidopsis thaliana), Cri (Ceratopteris richardii), Cpl (Craterostigma plantagineum), Dca (Daucus carota), Gma (Glycine max), Han (Helianthus annuus), Les (Lycopersicon esculentum), Osa (Oryza sativa), Psp. (Phalaenopsis sp.), Ppa (Physcomitrella patens), Pbr (Pimpinella brachycarpa), Par (Prunus armeniaca), Pta (Pinus taeda) and Zma (Zea mays). Symbols after the abbreviated species names indicate the higher classification: eudicots (black circles), monocots (hatched circles), gymnosperms (triangles), ferns (open circles), and mosses (double circles). The brackets on the right indicate the different subfamilies of the HD-Zip gene family

 
The HD-Zip family has been classified into four subfamilies, namely, HD-Zip I, II, III, and IV, based on amino acid sequences (Sessa, Morelli, and Ruberti 1993Citation ; Meijer et al. 1997Citation ; Aso et al. 1999Citation ). Pphb1, Pphb2, Pphb5, Pphb6, Pphb7, Pphb8, and Pphb9 form a clade with 93% local bootstrap support which is included in the HD-Zip I subfamily. In this subfamily, seven additional amino acid residues in the latter half of the leucine zipper motif distinguish the fern C. richardii HD-Zip I genes Crhb4, Crhb5, Crhb6, Crhb7, Crhb8, and Crhb11 (Aso et al. 1999Citation ). The Pphb genes do not have these additional residues and are similar to angiosperm HD- Zip I genes (fig. 2 ).

Pphb4 clusters in the HD-Zip II subfamily, whose monophyly is supported with high local bootstrap probability (91%). Pphb3 is sister to the HD-Zip II subfamily with 87% local bootstrap support. Pphb10 clusters in the HD-Zip III subfamily, whose monophyly has 99% local bootstrap support. No gene that clusters with the HD- Zip IV subfamily was found in this study.

Difference in Evolutionary Rates Between the HD-Zip I and the HD-Zip II Subfamilies
Six species pairs had ortholog pairs in both the HD- Zip I and the HD-Zip II subfamilies. The compared ortholog pairs and the result of comparisons are shown in tables 1, 2, 3, 4, 5, and 6 . In comparisons of distances between ortholog pairs of a eudicot (Arabidopsis, Glycine, or Lycopersicon) and a monocot (Oryza), every HD-Zip I ortholog pair had a longer distance than any HD-Zip II ortholog pair. In most comparisons, >95% of differences in the bootstrap samples were positive, and the minimum bootstrap proportion was 79%. On the other hand, some comparisons of species pairs between eudicots were negative but not significantly different from 0.


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Table 1 The Differences in Evolutionary Distances from the HD-Zip I Gene to the HD-Zip II Gene (Arabidopsis and Oryza orthologous pairs)

 

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Table 2 The Differences in Evolutionary Distances from the HD-Zip I gene to the HD-Zip II Gene (Glycine and Oryza orthologous pairs)

 

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Table 3 The Differences in Evolutionary Distances from the HD- Zip I Gene to the HD-Zip II Gene (Lycopersicon and Oryza orthologous pairs)

 

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Table 4 The Differences in Evolutionary Distances from the HD-Zip I Gene to the HD-Zip II Gene (Arabidopsis and Glycine orthologous pairs)

 

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Table 5 The Differences in Evolutionary Distances from the HD-Zip I Gene to the HD-Zip II Gene (Arabidopsis and Lycopersicon orthologous pairs)

 

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Table 6 The Differences in Evolutionary Distances from the HD- Zip I Gene to the HD-Zip II Gene (Glycine and Lycopersicon orthologous pairs)

 
The Leucine Zipper Motif of Pphb Genes
The leucine zipper motif consists of several heptad repeats. Each site in a heptad repeat is assigned an alphabetical letter from a to g (fig. 2 ). The leucine residues are usually found in the a and d sites and play important roles in protein dimerization (Hu et al. 1990Citation ; Gonzalez et al. 1997Citation ).

In the angiosperm HD-Zip I genes, the a1 and d1 sites are mainly occupied by threonine and leucine, respectively. In the fern HD-Zip I genes, the a1 site is occupied by asparagine instead of threonine, and the d1 site is variable and occupied by leucine, valine, or isoleucine (Aso et al. 1999Citation ). The a1 sites of the Pphb genes in the HD-Zip I subfamily are all threonine, except for Pphb9, which has isoleucine. Leucine residues occupy the d1 sites as observed in angiosperm HD-Zip I genes.

The a1 and d1 sites of HD-Zip II genes are fixed with leucine and threonine, respectively. As in the angiosperms, the a1 and d1 sites of Pphb4 are leucine and threonine, respectively. The a1 and d1 sites of Pphb3 are alanine and asparagine, respectively, unlike any other HD-Zip genes.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic Relationships Among HD-Zip Subfamilies of P. patens
Previously, HD-Zip genes were known only in vascular plants and not in other multicellular organisms. In this study, we isolated 10 HD-Zip genes from the moss P. patens. This is the first report of HD-Zip genes isolated from nonvascular green plants, suggesting that HD-Zip genes originated before the divergence of the mosses and vascular plants. Previous studies suggested that the HD-Zip gene family divided into four subfamilies, HD-Zip I, II, III, and IV, before the seed plants and ferns split (Aso et al. 1999Citation ). The HD-Zip gene tree in figure 3 indicates that the divergence of the four subfamilies occurred before the split of the mosses. In Aso et al. (1999)Citation , the HD-Zip III and IV subfamilies were consistently mislabeled, such that the HD-Zip III subfamily was called the HD-Zip IV subfamily and vice versa. The nomenclature used in this paper is consistent with all publications prior to Aso et al. (1999)Citation . Pphb3 is sister to the HD-Zip II subfamily and has two unique characters: (1) the long distance separating Pphb3 from its sister, the HD-Zip II subfamily, and (2) the divergence of amino acid residues in the otherwise conserved a1 and d1 sites of the leucine zipper motif. Pphb3 may constitute a new subfamily, which will be confirmed by further screening of the HD-Zip genes in other plants.

The fact that the mosses have HD-Zip I and II genes is not surprising, because their roles in angiosperms (e.g., light, ABA, and auxin signal transduction networks) are likely general to all plants, including mosses. A phytochrome gene which encodes a photosensor pigment of plants has been cloned in P. patens (Kolukisaoglu et al. 1993Citation ), and some phototropic responses that should be caused by the light signal transduction network have also been reported in mosses (Russell et al. 1998Citation ). The desiccation stress response networks mediated by ABA, which involve angiosperm HD-Zip I or II genes, are likely conserved between P. patens and angiosperms (Knight et al. 1995Citation ). Auxin is an important developmental regulator in both angiosperms and mosses (reviewed in Cove 1992Citation ), although its roles in development are different in the two because of their developmental and morphological differences. It is unclear whether these plants use similar cascades for light-, ABA-, and auxin-mediated networks, and further characterization of P. patens HD-Zip I and II gene functions and comparison with angiosperm HD-Zip genes are necessary.

Most angiosperm HD-Zip III genes are involved in the differentiation of vascular tissue (Baima et al. 1995Citation ; Zhong and Ye 1999Citation ). Some mosses, including P. patens, have water-conducting hydroids and nutrient-conducting leptoids in the gametophyte similar to the tracheids and sieve elements of vascular plants, respectively (Bell 1992Citation , pp.118–120). However, the homologies of these cells are ambiguous, because in vascular plants, tracheids and sieve elements are formed in the sporophyte generation. Furthermore, the cell wall thickening common to tracheids does not occur in hydroids, although the leptoids are similar to sieve elements at the ultrastructural level (Schofield and Hébant 1984Citation ). Determining the function of Pphb10, the moss HD-Zip III gene, may give insights into the evolution of vascular tissue in land plants.

Differences in Evolutionary Rates Between the HD- Zip I and the HD-Zip II Subfamilies
The amino acid residues in the homeodomain and the leucine zipper motif of the HD-Zip I subfamily appear less conserved than those of the HD-Zip II subfamily in the alignment (fig. 2 ). Chan et al. (1998)Citation pointed out the possibility that the evolutionary rate was higher in the former subfamily than in the latter. Comparison of the evolutionary distances for the ortholog pairs of eudicots and rice indicated that HD-Zip I genes evolved faster than HD-Zip II genes after the split of eudicots and monocots (tables 1–3 ). Within eudicots, the differences are not clear, perhaps because the difference has not accumulated enough to be significant (table 4–6 ). Some metazoan homeobox genes have been reported to function as a complex with other cofactors that regulate the homeobox gene (Mann and Chan 1996Citation ; Bürglin 1998Citation ; González-Crespo et al. 1998Citation ; Mann and Affolter 1998Citation ). The leucine zipper motif also plays a role in protein-protein interaction (O'Shea, Rutkowski, and Kim 1989Citation ). HD-Zip II genes may evolve slowly because HD- Zip II genes interact with cofactors that regulate the function of HD-Zip II genes strictly, while HD-Zip I genes may evolve faster because these genes do not interact strictly with cofactors and have relaxed specificity. In animals, helix 3 of the homeodomain is essential for DNA recognition (Kissinger et al. 1990Citation ), while helices 1 and 2 are involved in less stringent interactions with other proteins (Chan et al. 1994Citation ; Zappavigna, Sartori, and Mavilio 1994Citation ; Grueneberg et al. 1995Citation ). Figure 2 shows that the amino acid residues of helix 3 of HD- Zip I and II genes are highly conserved throughout HD- Zip I and II genes, while helices 1 and 2 of HD-Zip I genes have more variety than those of HD-Zip II genes. This suggests that a difference in the protein-protein interactions of the HD-Zip I and II subfamilies may be one of the reasons for the difference in their respective evolutionary rates.

Some HD-Zip genes are reported to dimerize via the leucine zipper motif and bind DNA in vitro (Sessa, Morelli, and Ruberti 1993Citation ; Gonzalez et al. 1997Citation ; Sessa et al. 1998Citation ). The a1 and d1 sites of the leucine zipper motif are important for determining the dimerization partner (Gonzalez et al. 1997Citation ). Amino acids in the a1 and d1 sites of HD-Zip I genes vary, while those of HD- Zip II genes are always leucine and threonine, respectively. Given that the amino acids of HD-Zip II genes are highly conserved, these genes likely select their dimerization partner more strictly than do HD-Zip I genes, perhaps to maintain specificity of pairing. Further studies on the protein-protein interactions of HD-Zip genes are required to test this hypothesis. The functions of those genes that have not been well characterized must also be studied, especially in additional nonvascular plants. Physcomitrella patens may be an ideal model with which to analyze gene functions, since it is amenable to transgenic methods (Cove, Knight, and Lamparter 1997Citation ; Schaefer and Zrÿd 1997Citation ). Such studies will help to elucidate the evolution of HD-Zip genes in land plants.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank two anonymous reviewers for their valuable comments on the manuscript, and Futamura Chemical Industries Co., Ltd., for providing the cellophane used to culture P. patens. The computations were done on a SUN Enterprize 3000 or an SGI Origin 2000 in the Computer Lab of NIBB. The NIBB Center for Analytical Instruments provided sequence facilities. T.N. is a research fellow of the Japan Society for the Promotion of Science. This research was partly supported by grants from the Ministry of Education, Science, Culture, and Sports, Japan (M.H., M.K., T.N.), and the Japan Society for the Promotion of Science (M.H., M.K., T.N.). K.S. and T.N. both contributed equally to this work.


    Footnotes
 
Elizabeth Kellogg, Reviewing Editor

1 Keywords: Physcomitrella moss homeobox leucine zipper molecular evolution gene family Back

2 Address for correspondence and reprints: Mitsuyasu Hasebe, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan. mhasebe{at}nibb.ac.jp Back


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 Introduction
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Accepted for publication November 14, 2000.