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.     

Classification of the genes involved in the two-component system of the moss Physcomitrella patens

Physcomitrella patens, belonging to bryopsida, is a basal lineage of land plants. To gain insight into the diversification of the two-component system (TCS), which is widely conserved from prokaryotes to eukaryotes, we compiled TCS-associated genes by employing P. patens genome databases. The moss has a set of His-kinases (HKs), including homologs of the cytokinin- and ethylene-receptors in seed plants. In addition, it has a number of coding-sequences specifying unique HKs. We found evidence that a putative cytokinin-receptor HK in P. patans serves as a sensor for this hormone, and that the HK activity of a putative ethylene-receptor homolog is regulated by ethylene, as observed for Arabidopsis thaliana.     

Polyphenol oxidases in Physcomitrella: functional PPO1 knockout modulates cytokinin-dependent developmentin the moss Physcomitrella patens

Polyphenol oxidases (PPOs) are copper-binding enzymes of the plant secondary metabolism that oxidize polyphenols to quinones. Although PPOs are nearly ubiquitous in seed plants, knowledge on their evolution and function in other plant groups is missing. This study reports on the PPO gene family in the moss Physcomitrella patens (Hedw.) B.S.G. asan example for an early divergent plant. The P. patens PPO multigene family comprises 13 paralogues. Phylogenetic analyses suggest that plant PPOs evolved with the colonization of land and that PPO duplications within the monophyletic P. patens paralogue clade occurred after the separation of the moss and seed plant lineages. PPO functionality was demonstrated for recombinant PPO6. P. patens was analysed for phenolic compounds and six substances were detected intracellularly by LC-MS analysis: 4-hydroxybenzoic acid, p-cumaric acid, protocatechuic acid, salicylic acid, caffeic acid, and an ester of caffeic acid. Targeted PPO1 knockout (d|ppo1) plants were generated and plants lacking PPO1 exhibited only ~30% of the wild-type PPO activity in the culture medium, thus suggesting extracellular localization of PPO1, which is in contrast to the mostly plastidic PPO localization in seed plants. Further, d|ppo1 lines formed significantly more gametophores with a reduced areal plant size, which could be related to an increase of endogenously produced cytokinins and indicates an impact of PPO1 on plant development. d|ppo1 plants were less tolerant towards applied 4-methylcatechol compared to the wild type, which suggests a role of extracellular PPO1 in establishing appropriate conditions by the removal of inhibitory extracellular phenolic compounds.     

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.     

基因组学技术大发展助力园艺植物研究取得新进展

园艺植物包括花卉、蔬菜、果树、部分瓜类(如西瓜(Citrullus lanatus)和甜瓜(Cucumis melo))和茶树(Camellia sinensis),在植物分类上涉及大量物种。园艺植物的基因组学和遗传学研究具有重要的理论价值和经济意义。基因组测序技术及相关生物信息学工具的发展为园艺植物基因组和分子生物学研究注入了新的活力。睡莲是一种重要的花卉植物,除了具有观赏价值,其进化地位也非常特殊,属于一种早期被子植物类群。最近,蓝星睡莲(N.colorata)的高质量基因组图谱绘制完成。通过系统分析和比较睡莲基因组与其它被子植物的基因组,研究者阐明了睡莲的进化位置及相关进化事件。所获得的高质量基因组序列将有助于园艺植物研究者开展深入的分子遗传学研究,鉴定到控制和调控花器官、花色花香及品质等众多性状的功能基因,从而推动基础研究的快速发展和加快新品种创制。     

The glycosyltransferase repertoire of the spikemoss Selaginella moellendorffii and a comparative study of its cell wall

Spike mosses are among the most basal vascular plants, and one species, Selaginella moellendorffii, was recently selected for full genome sequencing by the Joint Genome Institute (JGI). Glycosyltransferases (GTs) are involved in many aspects of a plant life, including cell wall biosynthesis, protein glycosylation, primary and secondary metabolism. Here, we present a comparative study of the S. moellendorffii genome across 92 GT families and an additional family (DUF266) likely to include GTs. The study encompasses the moss Physcomitrella patens, a non-vascular land plant, while rice and Arabidopsis represent commelinid and non-commelinid seed plants. Analysis of the subset of GT-families particularly relevant to cell wall polysaccharide biosynthesis was complemented by a detailed analysis of S. moellendorffii cell walls. The S. moellendorffii cell wall contains many of the same components as seed plant cell walls, but appears to differ somewhat in its detailed architecture. The S. moellendorffii genome encodes fewer GTs (287 GTs including DUF266s) than the reference genomes. In a few families, notably GT51 and GT78, S. moellendorffii GTs have no higher plant orthologs, but in most families S. moellendorffii GTs have clear orthologies with Arabidopsis and rice. A gene naming convention of GTs is proposed which takes orthologies and GT-family membership into account. The evolutionary significance of apparently modern and ancient traits in S. moellendorffii is discussed, as is its use as a reference organism for functional annotation of GTs.     

Plant expansins are a complex multigene family with an ancient evolutionary origin

Expansins are a group of extracellular proteins that directly modify the mechanical properties of plant cell walls, leading to turgor-driven cell extension. Within the completely sequenced Arabidopsis genome, we identified 38 expansin sequences that fall into three discrete subfamilies. Based on phylogenetic analysis and shared intron patterns, we propose a new, systematic nomenclature of Arabidopsis expansins. Further phylogenetic analysis, including expansin sequences found here in monocots, pine (Pinus radiata, Pinus taeda), fern (Regnellidium diphyllum, Marsilea quadrifolia), and moss (Physcomitrella patens) indicate that the three plant expansin subfamilies arose and began diversifying very early in, if not before, colonization of land by plants. Closely related "expansin-like" sequences were also identified in the social amoeba, Dictyostelium discoidium, suggesting that these wall-modifying proteins have a very deep evolutionary origin.     

Evolutionary crossroads in developmental biology: Physcomitrella patens

The moss Physcomitrella patens has recently emerged as a powerful genetically tractable model plant system. As a member of the bryophytes, P. patens provides a unique opportunity to study the evolution of a myriad of plant traits, such as polarized cell growth, gametophyte-to-sporophyte transitions, and sperm-to-pollen transition. The availability of a complete genome sequence, together with the ability to perform gene targeting efficiently in P. patens has spurred a flurry of elegant reverse genetic studies in this plant model that address a variety of key questions in plant developmental biology.     

An evolutionary view of functional diversity in family 1 glycosyltransferases

Glycosyltransferases (GTs) (EC 2.4.x.y) catalyze the transfer of sugar moieties to a wide range of acceptor molecules, such as sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules, including plant secondary metabolites. These enzymes can be classified into at least 92 families, of which family 1 glycosyltransferases (GT1), often referred to as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom. To understand how UGTs expanded in both number and function during evolution of land plants, we screened genome sequences from six plants (Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Arabidopsis lyrata) for the presence of a conserved UGT protein domain. Phylogenetic analyses of the UGT genes revealed a significant expansion of UGTs, with lineage specificity and a higher duplication rate in vascular plants after the divergence of Physcomitrella. The UGTs from the six species fell into 24 orthologous groups that contained genes derived from the common ancestor of these six species. Some orthologous groups contained multiple UGT families with known functions, suggesting that UGTs discriminate compounds as substrates in a lineage-specific manner. Orthologous groups containing only a single UGT family tend to play a crucial role in plants, suggesting that such UGT families may have not expanded because of evolutionary constraints.     

小立碗藓WRKY基因家族生物信息学分析

WRKY作为最先在植物中发现的转录因子,在植物生长发育等过程中发挥重要作用。为了更好地研究小立碗藓WRKY蛋白的结构与功能,该文以Pfam数据库中WRKY基因家族数据(登录号为PF03106)为材料,分析了小立碗藓(Physcomitrella patens)WRKY基因家族成员的理化性质、蛋白质的二级结构预测、染色体定位、内外显子分布及系统进化关系。结果表明:(1)小立碗藓WRKY基因家族成员共有38个基因,根据WRKY保守结构域个数和锌指结构类型分成Ⅰ、Ⅱ两大类,不含第Ⅲ类(锌指结构为C2HC型),其中部分基因WRKY保守结构域发生变异。(2)WRKY蛋白氨基酸长度在216~775 aa之间、相对分子质量在24.5~82.8 kDa之间,亚细胞定位显示WRKY家族成员蛋白质定位于细胞核中。(3)WRKY蛋白的二级结构以α-螺旋、延伸链、β-转角、无规卷曲四种构成元件构成,除PpWRKY11(α-螺旋为主)外,其余无规卷曲占比高达70%。(4)与拟南芥的系统进化关系表明,植物在进化过程中WRKY家族成员的数目与进化方式发生改变,WRKY基因家族成员外显子的个数为3~7个。(5)小立碗藓WRKY基因家族成员无规则分散于21条染色体上,并未形成基因簇。该研究通过分析WRKY基因家族的基本结构与性质,能为后续深入研究WRKY转录因子的功能奠定基础。     

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.     

Gene targeting in Physcomitrella patens

Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Sequencing programs and microbiological molecular genetic approaches are now being developed to unravel the precise function of plant genes. Physcomitrella patens, as the new 'green yeast', might well become a major tool for functional genomic studies of multicellular eukaryotes.     

Characterization of a PDK1 homologue from the moss Physcomitrella patens

The serine/threonine protein kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a highly conserved eukaryotic kinase that is a central regulator of many AGC kinase subfamily members. Through its regulation of AGC kinases, PDK1 controls many basic cellular processes, from translation to cell survival. While many of these PDK1-regulated processes are conserved across kingdoms, it is not well understood how PDK1 may have evolved within kingdoms. In order to better understand PDK1 evolution within plants, we have isolated and characterized the PDK1 gene from the moss Physcomitrella patens (PpPDK1), a nonvascular representative of early land plants. PpPDK1 is similar to other plant PDK1s in that it can functionally complement a yeast PDK1 knockout line. However, unlike PDK1 from other plants, the P. patens PDK1 protein does not bind phospholipids due to a lack of the lipid-binding pleckstrin homology domain, which is used for lipid-mediated regulation of PDK1 activity. Sequence analysis of several PDK1 proteins suggests that lipid regulation of PDK1 may not commonly occur in algae and nonvascular land plants. PpPDK1 can phosphorylate AGC kinase substrates from tomato (Solanum lycopersicum) and P. patens at the predicted PDK1 phosphorylation site, indicating that the PpPDK1 substrate phosphorylation site is conserved with higher plants. We have also identified residues within the PpPDK1 kinase domain that affect kinase activity and show that a mutant with highly reduced kinase activity can still confer cell viability in both yeast and P. patens. These studies lay the foundation for further analysis of the evolution of PDK1 within plants.     

Green algae to land plants: An evolutionary transition

Studies focused upon the evolutionary transition from ancestral green algae to the earliest land plants are important from a range of ecological, molecular and evolutionary perspectives. A substantial suite of ultrastructural, biochemical and molecular data supports the concept that land plants (embryophytes) are monophyletically derived from an ancestral charophycean alga. However, the details of phylogenetic branching patterns linking extant charophytes and seedless embryophytes are currently unclear. Moreover, the fossil record has so far been mute regarding the algae-land plant transition. Nevertheless, an accurate reflection of major evolutionary events in the history of the earliest land plants can be obtained by comparative paleontological-neontological studies, and comparative molecular, cellular and developmental investigations of extant charophytes and bryophytes. This review focuses upon research progress toward understanding three clade-specific adaptations that were important in the successful colonization of land by plants: the histogenetic apical meristem, the matrotrophic embryo, and decay-resistant cell wall polymers.     

Intron loss mediated structural dynamics and functional differentiation of the polygalacturonase gene family in land plants

The polygalacturonase (PG) gene family is one of the largest gene families in plants. PGs are involved in various plant development steps. The evolutionary processes accounting for the functional divergence and the specialized functions of PGs in land plants are unclear. Whole sets of PG genes were retrieved from the genome web sites of model organisms in algae and land plants. The number of PG genes was expanded by lineage-specific manner with the biological complexity of the organism. Differentiation of PGs was related with phylogenetic hierarchy such as presence of rhamno-PGs from algae to plants, endo- and exo-PGs in land plants, exo-PGs in flowering plants. Gene structure analysis revealed that land plant PG genes resulted from differential intron gain and loss, with the latter event predominating. Differential intron losses partitioned the PGs into separate clades to be expressed differentially during plant development. Intron position and phase were not conserved between PGs of algae and land plants but conserved among PG genes of land plants from moss to vascular plants, indicating that the current introns in the PGs in land plants appeared after the split between unicellular algae and multicelluar land plants. The results demonstrate that the functional divergence and differentiation of PGs in land plants is attributable to intron losses.