The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion

ABSTRACT: BACKGROUND: Plant polyphenol oxidases (PPOs) are enzymes that typically use molecular oxygen to oxidize ortho-diphenols to ortho-quinones. These commonly cause browning reactions following tissue damage, and may be important in plant defense. Some PPOs function as hydroxylases or in cross-linking reactions, but in most plants their physiological roles are not known. To better understand the importance of PPOs in the plant kingdom, we surveyed PPO gene families in 25 sequenced genomes from chlorophytes, bryophytes, lycophytes, and flowering plants. The PPO genes were then analyzed in silico for gene structure, phylogenetic relationships, and targeting signals. RESULTS: Many previously uncharacterized PPO genes were uncovered. The moss, Physcomitrella patens, contained 13 PPO genes and Selaginella moellendorffii (spike moss) and Glycine max (soybean) each had 11 genes. Populus trichocarpa (poplar) contained a highly diversified gene family with 11 PPO genes, but several flowering plants had only a single PPO gene. By contrast, no PPO-like sequences were identified in several chlorophyte (green algae) genomes or Arabidopsis (A. lyrata and A. thaliana). We found that many PPOs contained one or two introns often near the 3' terminus. Furthermore, N-terminal amino acid sequence analysis using ChloroP and TargetP 1.1 predicted that several putative PPOs are synthesized via the secretory pathway, a unique finding as most PPOs are predicted to be chloroplast proteins. Phylogenetic reconstruction of these sequences revealed that large PPO gene repertoires in some species are mostly a consequence of independent bursts of gene duplication, while the lineage leading to Arabidopsis must have lost all PPO genes. CONCLUSION: Our survey identified PPOs in gene families of varying sizes in all land plants except in the genus Arabidopsis. While we found variation in intron numbers and positions, overall PPO gene structure is congruent with the phylogenetic relationships based on primary sequence data. The dynamic nature of this gene family differentiates PPO from other oxidative enzymes, and is consistent with a protein important for a diversity of functions relating to environmental adaptation.     

Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, flavonoids have played an important role as UV protective pigments in plants. Flavonoids occur in liverworts and mosses, and the first committed step in the flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Although higher plant CHSs have been extensively studied, little information is available on the enzymes from bryophytes. Here we report the cloning and characterization of CHS from the moss, Physcomitrella patens. Taking advantage of the available P. patens EST sequences, a CHS (PpCHS) was cloned from the gametophores of P. patens, and heterologously expressed in Escherichia coli. PpCHS exhibited similar kinetic properties and substrate preference profile to those of higher plant CHS. p-Coumaroyl-CoA was the most preferred substrate, suggesting that PpCHS is a naringenin chalcone producing CHS. Consistent with the evolutionary position of the moss, phylogenetic analysis placed PpCHS at the base of the plant CHS clade, next to the microorganism CHS-like gene products. Therefore, PpCHS likely represents a modern day version of one of the oldest CHSs that appeared on earth. Further, sequence analysis of the P. patens EST and genome databases revealed the presence of a CHS multigene family in the moss as well as the 3'-end heterogeneity of a CHS gene. Of the 19 putative CHS genes, 10 genes are expressed and have corresponding ESTs in the databases. A possibility of the functional divergence of the multiple CHS genes in the moss is discussed.     

小立碗藓（Physcomitrella patens）植物microRNA结合靶位预测

利用857条植物miRNA序列对27546条小立碗藓mRNA序列进行搜索,预测出162个植物miRNA家族在小立碗藓中存在结合靶位。miRNA结合靶位数目和miRNA协同作用网络分析结果同时显示,miR482和miR1168在小立碗藓中结合靶位多、协同作用广,提示它们对于小立碗藓可能具有重要生物学功能。52个菜茵衣藻特有的miRNA被预测在小立碗藓中存在结合靶位,显示小立碗藓在从藻类向种子植物进化过程中处在独特演化位置。     

Exploring plant biodiversity: the Physcomitrella genome and beyond

For decades, plant molecular biology has focused on only a few angiosperm species. Recently, the approximately 500mega base pairs (Mb) of the haploid Physcomitrella patens genome were sequenced and annotated. Mosses such as P. patens occupy a key evolutionary position halfway between green algae and flowering plants. This draft genome, in comparison to existing genome data from other plants, allows evolutionary insights into the conquest of land by plants and the molecular biodiversity that land plants exhibit. As a model organism, P. patens provides a well-developed molecular toolbox, including efficient gene targeting in combination with the morphologically simple moss tissues. We describe current as well as future tools for P. patens research and the prospects they offer for plant research in general.     

Characterization of the PHO1 gene family and the responses to phosphate deficiency of Physcomitrella patens

PHO1 was previously identified in Arabidopsis (Arabidopsis thaliana) as a protein involved in loading inorganic phosphate (Pi) into the xylem of roots and its expression was associated with the vascular cylinder. Seven genes homologous to AtPHO1 (PpPHO1;1-PpPHO1;7) have been identified in the moss Physcomitrella patens. The corresponding proteins harbor an SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the conserved C-terminal hydrophobic portion, both common features of the plant PHO1 family. Northern-blot analysis showed distinct expression patterns for the PpPHO1 genes, both at the tissue level and in response to phosphate deficiency. Transgenic P. patens expressing the beta-glucuronidase reporter gene under three different PpPHO1 promoters revealed distinct expression profiles in various tissues. Expression of PpPHO1;1 and PpPHO1;7 was specifically induced by Pi starvation. P. patens homologs to the Arabidopsis PHT1, DGD2, SQD1, and APS1 genes also responded to Pi deficiency by increased mRNA levels. Morphological changes associated with Pi deficiency included elongation of caulonemata with inhibition of the formation of side branches, resulting in colonies with greater diameter, but reduced mass compared to Pi-sufficient plants. Under Pi-deficient conditions, P. patens also increased the synthesis of ribonucleases and of an acid phosphatase, and increased the ratio of sulfolipids over phospholipids. These results indicate that P. patens and higher plants share some common strategies to adapt to Pi deficiency, although morphological changes are distinct, and that the PHO1 proteins are well conserved in bryophyte despite the lack of a developed vascular system.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.     

Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.     

A novel plant major intrinsic protein in Physcomitrella patens most similar to bacterial glycerol channels

A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP.     

Ancestry of plant MADS-box genes revealed by bryophyte (Physcomitrella patens) homologues

Three MADS-box cDNA clones and two corresponding genomic sequences (gDNAs) have been isolated from the bryophyte Physcomitrella patens and sequenced. Our findings indicate that the genes may be expressed in a tissue- or age-specific manner, and that expression of one of them is regulated by an alternative splicing mechanism. Conceptual translation of the clones reveals that the encoded MADS-domain proteins have the typical plant-domain pattern (MIKC). Additionally, there is a high degree of conservation of intron number and positions between angiosperm MADS-box genes and the moss loci. These observations confirm the homology of moss and higher plant MADS-box genes. We conclude that the MIKC pattern evolved in MADS-box genes after the separation of the plant lineage from that of fungi and animals, and that it must have been present in the common ancestor of mosses, ferns and seed plants. Therefore it evolved at least 400 million yr ago. Phylogenetic analysis of a large subset of the sequenced plant MADS-box genes, incorporating those from P. patens , indicates that the bryophyte genes are not orthologues of spermatophyte genes belonging to any of the well recognized higher plant gene subfamilies. This conclusion accords well with reports that the known fern MADS-box genes also comprise subfamilies distinct from those of higher plants. Therefore we tentatively propose that the gene duplication and diversification events that created the MADS-box gene subfamilies, discernible in extant angiosperm and other spermatophyte groups, occurred after separation of the moss and fern lineages from the lineage which produced the higher plants.     

Identification and characterization of cDNAs encoding pentatricopeptide repeat proteins in the basal land plant, the moss Physcomitrella patens

A large gene family encoding proteins with a pentatricopeptide repeat (PPR) motif exists in flowering plants but not in algae, fungi, or animals. This suggests that PPR protein genes expanded vastly during the evolution of the land plants. To investigate this possibility, we analysed PPR protein genes in the basal land plant, the moss Physcomitrella patens. An extensive survey of the Physcomitrella expressed sequence tag (EST) databases revealed 36 ESTs encoding PPR proteins. This indicates that a large gene family of PPR proteins originated before the divergence of the vascular plant and moss lineages. We also characterized five full-length cDNAs encoding PPR proteins, designated PPR513-10, PPR566-6, PPR868-14, PPR986-12, and PPR423-6. Intracellular localization analysis demonstrated two PPR proteins in chloroplasts (cp), whereas the cellular localization of the other three PPR proteins is unclear. The genes of the cp-localized PPR513-10 and PPR566-6 were expressed differentially in protonemata grown under different light-dark conditions, suggesting they have distinctive functions in cp. This is the first report and analysis of genes encoding PPR proteins in bryophytes.     

苔藓植物小立碗藓，功能基因组学研究新的模式系统

苔藓植物具有相对简单的发育模式,单倍体的配子体在其生活史中占主导地位,作为研究植物生物学过程的模式系统具有诸多的优越性。苔藓植物小立碗藓能够高效地通过同源重组的方式将外源核酸整合到其核DNA,这就使得基因打靶在此物种中就像在小鼠胚胎干细胞和酵母中一样成为一个非常便利的技术。另外由于小立碗藓与高等植物在生物特征上有很大相似之处加之其有其他诸多优越性,它有望成为一个诱人的植物生物学和功能基因组学研究的模式系统。Abstract：The potential of moss as a model system to study plant biological process is associated with their relatively simple developmental pattern and the dominance of the haploid gametophyte in the life cycle. The moss Physcomitrella patens exhibits a very high rate of homologous recombination in its nuclear DNA, making gene targeting approaches in this plant as convenient as in yeast or in ES cells of mice. Sharing many biological features with higher plants and having many other advantages, the moss Physcomitrella patens will be an attractive model system for plant biology and functional genome analysis.     

Heterologous expression and functional characterization of a Physcomitrella Pseudo response regulator homolog, PpPRR2, in Arabidopsis

Physcomitrella patens has four homologs of the pseudo-response regulator involved in the circadian clock mechanism in seed plants. To gain insight into their function, Arabidopsis transgenic lines misexpressing PpPRR2 were constructed. Phenotypic analysis of the transformants with reference to clock-related gene expression and photoperiodic responses revealed that heterologous expression of the moss PpPRR2 gene modifies the intrinsic mechanism underlying the circadian clock in Arabidopsis, suggesting that PpPRR2 serves as a clock component in P. patens.     

Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.