Revealing genetic diversity of tree peonies at micro-evolution level with hyper-variable chloroplast markers and floral traits

Key message

Highly variable regions of chloroplast genome were found to be useful in the detection of plant genetic diversity at micro-evolution level. Our methodology will improve understanding and conservation of plant diversity.

Abstract

Tree peonies are famous flowers with about 2,000 cultivars in the world, belonging to Paeonia sect. Moutan of the Paeoniaceae. They are traditionally classified based on flower forms and colors. Due to the limited number of DNA and morphological markers, and the existence of synonyms and homonyms, evaluation on genetic diversity of so many cultivars remains a challenge. In most cases, it is difficult and even impossible to discriminate tree peony cultivars when they are not in flower. In this study, single nucleotide polymorphism detected from the hyper-variable regions of chloroplast genome was employed to separate tree peony cultivars into different maternal lineages which can be expressed briefly by a nucleotide molecular formula. Our approach enabled a much higher resolution of cultivar identification and classification that has not been obtained before. The newly developed hyper-variable chloroplast markers, as an independent source of taxonomic characteristics, provided novel evidences and higher resolution ability that are helpful in building an effective classification system for evaluation, conservation, and utilization of the tree peony germplasm resources at cultivar level.     

Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle

The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12 h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria.     

GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.G protein-coupled receptor kinase-5 (GRK5)² is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs) (1, 2). We recently reported that increased soluble β-amyloid decreases membrane (functional) levels of GRK5 in vitro, and this membrane GRK5 deficiency occurs in vivo as well in an Alzheimer disease (AD) transgenic model (3) and in postmortem human AD brain samples (4). Moreover, the aged GRK5 knock-out (GRK5KO) mouse, which models this GRK5 deficiency in the absence of exogenous mutant human β-amyloid precursor protein (β-APP) or any other known AD-related genes (i.e. presenilins or tau), develops axonal defects and mild cholinergic degeneration with associated amnestic mild cognitive impairment (5). When Swedish mutant βAPP is overexpressed in the GRK5KO mice by cross-breeding with Swedish APP transgenic mice, the aged double mutant mice display significantly exaggerated brain inflammation (6). These accumulating data strongly suggest that GRK5 deficiency significantly contributes to AD pathogenesis, although the precise molecular mechanisms remain to be delineated.Mounting evidence indicates that the substrate spectrum of broadly expressed GRKs (i.e. GRK2/3/5/6) can significantly overlap for some receptors, suggesting that a lack of one of these members may have only a limited impact on GPCR regulation (2). On the other hand, compensation for loss of a particular GRK member by others in vivo can be incomplete or selective for other receptor types. For example, GRK2KO and GRK6KO mice have been shown to display selective impairments of adrenergic and dopaminergic receptor desensitization, respectively (7, 8). Findings from different GRK isoform-targeted animals strongly support the conclusion that although redundancy exists between GRK isoforms, each isoform has its own selective substrates; should one GRK be deficient or inactivated, desensitization of its selective substrates will be impaired (1). For GRK5 in particular, previous studies have demonstrated that GRK5KO mice display selectively impaired desensitization of muscarinic acetylcholine receptors (mAChRs) (9, 10).To date, five mAChR subtypes have been identified, with M1, M3, and M5 receptors being G_q/11-coupled, and M2 and M4 receptors being G_i/o-coupled (11). In hippocampal memory circuits, M2 receptor (M2R) is primarily a presynaptic autoreceptor that inhibits ACh release (12, 13), whereas M1R is postsynaptic and is believed to be critical in memory processes involving an interaction between the cerebral cortex and hippocampus (11). In AD, there is a selective loss of cholinergic neurons that leads to a cholinergic hypofunction, primarily a hypoactivity of postsynaptic nicotinic and M1 muscarinic receptors (14). GRK5KO mice, when challenged with nonselective muscarinic agonists, display augmented hypothermia, hypoactivity, tremor, and salivation, as well as antinociceptive changes (9). These behavioral changes are typical M2 and/or M4 receptor-mediated functions, according to the findings from muscarinic receptor subtype knock-out mice (11, 15). Therefore, GRK5 deficiency in vivo may selectively impair M2/M4R desensitization. If so, the resulting presynaptic M2/M4R hyperactivity would overly inhibit ACh release from cholinergic neurons and eventually compromise the learning and memory function. This study was undertaken to investigate the impact of GRK5 deficiency on ACh release and desensitization of mAChR subtypes using GRK5-deficient models both in vitro and in hippocampal slices from the GRK5KO mice.     

脉叶虎皮楠的生物碱成分研究

从脉叶虎皮楠(Daphniphyllum paxianum)的枝干中分离并鉴定了四个生物碱,分别为daphnilactone A(1)、daphnicyclidin D(2)、daphniphylline(3)、daphnicyclidin H(4).其中首次对化合物1的碳谱数据进行了归属.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.     

水盾草科系统位置评述

水盾草科（Cabombaceae）是双子叶植物。水盾草科包括2属：水盾草属（Cabomba Aublet．）和莼菜属（Brasenia Schreb．）。形态学研究显示水盾草科具有许多原始性状,而且在其适应水生环境的过程中经历了性状退化。水盾草科又被称为“古草本”。最新的分子系统发育研究显示,水盾草科是现存被子植物系统树基部ANITA类群的成员之一。但有关水盾草科的系统位置存在争议。被子植物的起源与早期分化一直是植物学家关注的热点。本文对该科系统位置的研究历史与现状进行评述。     

Sonneratia ovata Backer–A genetically depauperate mangrove species

It is expected that geographically widespread and outcrossing tree species would have high level of genetic variation. Three chloroplast regions and six nuclear genes were sequenced to assess the genetic variation of a species of mangroves, Sonneratia ovata, from China and Thailand. No nucleotide polymorphism was found in the three chloroplast regions and in the six nuclear genes from all of the four populations examined. The depauperation of polymorphism in S. ovata in comparison to moderate polymorphisms of other congeneric species is surprising, particularly considering high level of polymorphism in the past and relatively wide geographic distribution of the species. Since multiple independent loci were surveyed in this study, the most plausible explanation for our observation is that S. ovata has experienced severe demographic bottlenecks in the Pleistocene glaciation, followed by subsequent recolonization by sea currents in China and Southeast Asia regions. The lack of polymorphism may also be attributable to its small population size, despite its wide geographic distribution.