Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.     

Exploring the Mechanism of Physcomitrella patens Desiccation Tolerance through a Proteomic Strategy

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plant's mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.     

Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens

Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-¹⁴C]adenine to N⁶-[¹⁴C](Δ²-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N⁶-(Δ²-isopentenyl)adenine and N⁶-(Δ²-isopentenyl)adenosine monophosphate.     

The Nitric Oxide Production in the Moss Physcomitrella patens Is Mediated by Nitrate Reductase

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.     

Molecular Characterization of Three PRORP Proteins in the Moss Physcomitrella patens: Nuclear PRORP Protein Is Not Essential for Moss Viability

RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNA^Asp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA^Arg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA^Arg (CCG) and pre-tRNA^Arg (UCU) but they cleaved pre-tRNA^Arg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.     

糖尿病小型猪三磷酸腺苷结合盒转运体A1表达的变化

用贵州小香猪建立2型糖尿病动物模型,探讨糖尿病小型猪三磷酸腺苷结合盒转运体A1(ABCA1)表达的变化.采用高脂高蔗糖饲料喂养贵州小香猪,建立2型糖尿病动物模型.血浆总胆固醇、甘油三酯、高密度脂蛋白胆固醇和葡萄糖的浓度均用氧化酶法测定,血浆游离脂肪酸(FFA)用比色法测定, 采用逆转录-聚合酶链反应、蛋白质印迹和免疫组织化学分别检测ABCA1mRNA和蛋白质的表达.喂养6个月后,实验组与正常对照组比较,空腹血糖值明显升高;空腹胰岛素水平在头3个月轻度升高,在第6个月末其水平降低; 血清总胆固醇、甘油三酯和游离脂肪酸水平升高;肝组织、冠状动脉、肾组织ABCA1表达上调,同时观测到糖尿病小型猪肝组织LXRα表达上调.结果提示高脂高蔗糖饲料可引起小型猪脂质和糖代谢紊乱, 并导致肝组织、冠状动脉和肾组织ABCA1表达上调以及肝组织LXRα表达上调.     

Kinesins Are Indispensable for Interdigitation of Phragmoplast Microtubules in the Moss Physcomitrella patens

Microtubules form arrays with parallel and antiparallel bundles and function in various cellular processes, including subcellular transport and cell division. The antiparallel bundles in phragmoplasts, plant-unique microtubule arrays, are mostly unexplored and potentially offer new cellular insights. Here, we report that the Physcomitrella patens kinesins KINID1a and KINID1b (for kinesin for interdigitated microtubules 1a and 1b), which are specific to land plants and orthologous to Arabidopsis thaliana PAKRP2, are novel factors indispensable for the generation of interdigitated antiparallel microtubules in the phragmoplasts of the moss P. patens. KINID1a and KINID1b are predominantly localized to the putative interdigitated parts of antiparallel microtubules. This interdigitation disappeared in double-deletion mutants of both genes, indicating that both KINID1a and 1b are indispensable for interdigitation of the antiparallel microtubule array. Furthermore, cell plates formed by these phragmoplasts did not reach the plasma membrane in ∼20% of the mutant cells examined. We observed that in the double-deletion mutant lines, chloroplasts remained between the plasma membrane and the expanding margins of the cell plate, while chloroplasts were absent from the margins of the cell plates in the wild type. This suggests that the kinesins, the antiparallel microtubule bundles with interdigitation, or both are necessary for proper progression of cell wall expansion.     

Tagged Mutagenesis and Gene-trap in the Moss, Physcomitrella patens by Shuttle Mutagenesis

The moss, Physcomitrella patens has been used as a useful materialin many fields, because of its simple body plan, ease of genetargeting, and other reasons. Although many mutants have beenreported, no method to isolate the corresponding genes was reported.We developed a gene tagging and gene-trap system in P. patensby using the shuttle mutagenesis technique, which has been usedin the budding yeast. In 5264 tagged lines, 203 mutants withaltered developmental or morphological phenotypes were obtained.In 129 of 4757 gene-trap lines, ß-glucuronidase (GUS)activity was detected in some tissue. Although multiple copiesof a tag were detected in many tagged lines by Southern analyses,most copies are likely integrated at the same locus accordingto PCR analyses.     

转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展

：ABCC4、ABCC5是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和排泄等。ABCC4、ABCC5的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展进行综述。     

动脉粥样硬化小型猪三磷酸 腺苷结合盒转运体 A1 表达的变化

用贵州小香猪建立动脉粥样硬化动物模型,探讨动脉粥样硬化小型猪三磷酸腺苷结合盒转运体 A1(ABCA1) 表达的变化 . 采用血管内膜损伤法加高脂高胆固醇饲料喂养贵州小香猪,建立动脉粥样硬化动物模型 . 血浆总胆固醇、甘油三酯和高密度脂蛋白胆固醇的浓度均用氧化酶法测定,采用逆转录聚合酶链反应检测 ABCA1mRNA 水平,蛋白质印迹和免疫组织化学检测 ABCA1 蛋白质的表达 . 喂养 12 个月后,实验组与正常对照组比较,空腹血浆总胆固醇、甘油三酯和高密度脂蛋白胆固醇水平升高;实验组小型猪主动脉、髂动脉、颈总动脉和冠状动脉可见动脉粥样硬化斑块和脂质条纹;实验组小型猪肝组织、主动脉、小肠组织 ABCA1 表达上调 . 结果提示,采用血管内膜损伤法加高脂高胆固醇饲料喂养小型猪可建立动脉粥样硬化动物模型 . 动脉粥样硬化小型猪肝组织、主动脉和小肠组织 ABCA1 表达上调 .     

转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展

ABCC4、ABCC5 是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性 代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5 在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和 排泄等。ABCC4、ABCC5 的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5 与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就 转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展进行综述。     

ATP Requirement for Chloroplast Protein Import Is Set by the Km for ATP Hydrolysis of Stromal Hsp70 in Physcomitrella patens

The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the K_m for ATP and a decrease in the enzyme’s k_cat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the K_m-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the K_m for ATP hydrolysis of Hsp70 translated into an increased K_m for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone’s ATPase activity dominates the energetics of the reaction.     

三磷酸腺苷结合盒转运体A1在脑疾病发生发展中的作用

三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1,ABCA1)在脑组织中广泛表达,它将脑细胞内胆固醇转运给载脂蛋白E(apolipoprotein E,apoE)及载脂蛋白A1(apolipoprotein A1,apoA-Ⅰ)形成高密度脂蛋白(high density lipoprotein,HDL),从而调控脑内胆固醇平衡.研究表明,ABCA1与胆固醇代谢相关脑疾病存在密切联系,包括阿尔茨海默病(Alzheimer's disease,AD)、创伤性脑损伤(traumatic brain injury,TBI)及脑梗死.虽然近来在ABCA1与相关脑疾病的研究取得了一些进展,但仍存在许多问题尚未阐明.本文对ABCA1在各种相关脑疾病发生发展中的作用做一综述,期望为相关脑疾病的治疗寻找新的靶点和方法.     

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.     

Myosin XI Is Essential for Tip Growth in Physcomitrella patens

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.     

N-Glycosylation in the Moss Physcomitrella patens is Organized Similarly to that in Higher Plants

Abstract: Allergenicity of plant glycoproteins in humans may prevent the use of plants as production factories for pharmaceutically important proteins. The major difference between plant and mammalian N-glycans is the presence of xylosyl and α1,3-fucosyl residues in the former. In a first step towards "humanization" of the N-glycosylation pathway in the moss Physcomitrella patens, which could be an excellent system for industrial production of therapeutic proteins, we isolated the cDNAs and genes for N-acetylglucosaminyltransferase I (GNTI), α1,3-fucosyltransferase, and β1,2-xylosyltransferase. Sequence analysis revealed that all three proteins are homologous to their counterparts from higher plants, however, the conservation of the primary structure was only 35 - 45 %. The gene encoding the key enzyme of the pathway, gntI, was disrupted in P. patens by homologous recombination. Although the mutation of this gene in mouse or A. thaliana led to a significantly altered pattern of N-glycans, the glycosylation pattern in the gntI knockouts did not differ from that in wild-type moss and was identical to that in higher plants. Protein secretion, analysed in assays with recombinant human VEGF₁₂₁ protein, was not affected in the knockouts. We conclude from our findings that the N-glycosylation pathway in P. patens is identically organized to that in higher plants. However, P. patens probably possesses more than one isoform of GNTI which complicates a straightforward knockout. Therefore, and since complex type structures appear more desirable than oligomannosidic N-glycans, future modifications of the pathway should target α1,3-fucosyltransferase and/or β1,2-xylosyltransferase.     

Suppression of Repeat-Mediated Gross Mitochondrial Genome Rearrangements by RecA in the Moss Physcomitrella patens

RecA and its ubiquitous homologs are crucial components in homologous recombination. Besides their eukaryotic nuclear counterparts, plants characteristically possess several bacterial-type RecA proteins localized to chloroplasts and/or mitochondria, but their roles are poorly understood. Here, we analyzed the role of the only mitochondrial RecA in the moss Physcomitrella patens. Disruption of the P. patens mitochondrial recA gene RECA1 caused serious defects in plant growth and development and abnormal mitochondrial morphology. Analyses of mitochondrial DNA in disruptants revealed that frequent DNA rearrangements occurred at multiple loci. Structural analysis suggests that the rearrangements, which in some cases were associated with partial deletions and amplifications of mitochondrial DNA, were due to aberrant recombination between short (<100 bp) direct and inverted repeats in which the sequences were not always identical. Such repeats are abundant in the mitochondrial genome, and interestingly many are located in group II introns. These results suggest that RECA1 does not promote but rather suppresses recombination among short repeats scattered throughout the mitochondrial genome, thereby maintaining mitochondrial genome stability. We propose that RecA-mediated homologous recombination plays a crucial role in suppression of short repeat-mediated genome rearrangements in plant mitochondria.