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1.

Aims

This study investigated Cu uptake and accumulation as well as physiological and biochemical changes in grapevines grown in soils containing excess Cu.

Methods

The grapevines were collected during two productive cycles from three vineyards with increasing concentrations of Cu in the soil and at various growth stages, before and after the application of Cu-based fungicides. The Cu concentrations in the grapevine organs and the macronutrients and biochemical parameters in the leaf blades were analyzed.

Results

At close to the flowering stage of the grapevines, the concentration and content of Cu in the leaves were increased. However, the Cu concentrations in the roots, stem, shoots and bunches did not correlate with the metal concentrations in the soil. The application of Cu-based fungicides to the leaves increased the Cu concentrations in the shoots, leaves and rachis; however, the effect of the fungicides on the Cu concentration in the berries was not significant. The biochemical analyses of the leaf blades demonstrated symptoms of oxidative stress that correlated with the Cu concentrations in soil.

Conclusions

The increased availability of Cu in soil had a slight effect on the levels and accumulation of Cu in mature grapevines during the productive season and did not alter the nutritional status of the plant. However, increased Cu concentrations were observed in the leaves. The evidence of oxidative stress in the leaves correlated with the increased levels of Cu in soil.  相似文献   
2.
3.
High-throughput phenotyping is emerging as an important technology to dissect phenotypic components in plants. Efficient image processing and feature extraction are prerequisites to quantify plant growth and performance based on phenotypic traits. Issues include data management, image analysis, and result visualization of large-scale phenotypic data sets. Here, we present Integrated Analysis Platform (IAP), an open-source framework for high-throughput plant phenotyping. IAP provides user-friendly interfaces, and its core functions are highly adaptable. Our system supports image data transfer from different acquisition environments and large-scale image analysis for different plant species based on real-time imaging data obtained from different spectra. Due to the huge amount of data to manage, we utilized a common data structure for efficient storage and organization of data for both input data and result data. We implemented a block-based method for automated image processing to extract a representative list of plant phenotypic traits. We also provide tools for build-in data plotting and result export. For validation of IAP, we performed an example experiment that contains 33 maize (Zea mays ‘Fernandez’) plants, which were grown for 9 weeks in an automated greenhouse with nondestructive imaging. Subsequently, the image data were subjected to automated analysis with the maize pipeline implemented in our system. We found that the computed digital volume and number of leaves correlate with our manually measured data in high accuracy up to 0.98 and 0.95, respectively. In summary, IAP provides a multiple set of functionalities for import/export, management, and automated analysis of high-throughput plant phenotyping data, and its analysis results are highly reliable.Plant bioinformatics faces the challenge of integrating information from the related “omics” fields to elucidate the functional relationship between genotype and observed phenotype (Edwards and Batley, 2004), known as the genotype-phenotype map (Houle et al., 2010). One of the main obstacles is our currently limited ability of systemic depiction and quantification of plant phenotypes, representing the so-called phenotyping bottleneck phenomenon (Furbank and Tester, 2011). To get a comprehensive genotype-phenotype map, more accurate and precise phenotyping strategies are required to empower high-resolution linkage mapping and genome-wide association studies in order to uncover underlying genetic variants associated with complex phenotypic traits, which aim to improve the efficiency, effectiveness, and economy of cultivars in plant breeding (Cobb et al., 2013). In the era of phenomics, automatic high-throughput phenotyping in a noninvasive manner is applied to identify and quantify plant phenotypic traits. Plants are bred in fully automated greenhouses under predefined environmental conditions with controlled temperature, watering, and humidity. To meet the demand of data access, exchange, and sharing, several phenomics-related projects in the context of several consortia have been launched, such as the International Plant Phenotyping Network (http://www.plantphenomics.com/), the European Plant Phenotyping Network (http://www.plant-phenotyping-network.eu/), and the German Plant Phenotyping Network (http://www.dppn.de/).Thanks to the development of new imaging and transport systems, various automated or semiautomated high-throughput plant phenotyping systems are being developed and used to examine plant function and performance under controlled conditions. PHENOPSIS (Granier et al., 2006) is one of the pioneering platforms that was developed to dissect genotype-environment effects on plant growth in Arabidopsis (Arabidopsis thaliana). GROWSCREEN (Walter et al., 2007; Biskup et al., 2009; Jansen et al., 2009; Nagel et al., 2012) was designed for rapid optical phenotyping of different plant species with respect to different biological aspects. Other systems in the context of high-throughput phenotyping include Phenodyn/Phenoarch (Sadok et al., 2007), TraitMill (Reuzeau et al., 2005; Reuzeau, 2007), Phenoscope (Tisné et al., 2013), RootReader3D (Clark et al., 2011), GROW Map (http://www.fz-juelich.de/ibg/ibg-2/EN/methods_jppc/methods_node.html), and LemnaTec Scanalyzer 3D. These developments enable the phenotyping of specific organs (e.g. leaf, root, and shoot) or of whole plants. Some of them are even used for three-dimensional plant analysis (Clark et al., 2011). Consequently, several specific software applications (a comprehensive list can be found at http://www.phenomics.cn/links.php), such as HYPOTrace (Wang et al., 2009), HTPheno (Hartmann et al., 2011), LAMINA (Bylesjö et al., 2008), PhenoPhyte (Green et al., 2012), Rosette Tracker (De Vylder et al., 2012), LeafAnalyser (Weight et al., 2008), RootNav (Pound et al., 2013), SmartGrain (Tanabata et al., 2012), and LemnaGrid, were designed to extract a wide range of measurements, such as height/length, width, shape, projected area, digital volume, compactness, relative growth rate, and colorimetric analysis.The huge amount of generated image data from various phenotyping systems requires appropriate data management as well as an appropriate analytical framework for data interpretation (Fiorani and Schurr, 2013). However, most of the developed image-analysis tools are designed for a specific task, for specific plant species, or are not freely available to the research community. They lack flexibility in terms of needed adaptations to meet new analysis requirements. For example, it would be desirable that a system could handle imaging data from different sources (either from fully automated high-throughput phenotyping systems or from setups where images are acquired manually), different imaging modalities (fluorescence, near-infrared, and thermal imaging), and/or different species (wheat [Triticum aestivum], barley [Hordeum vulgare], maize [Zea mays], and Arabidopsis).In this work, we present Integrated Analysis Platform (IAP), a scalable open-source framework, for high-throughput plant phenotyping data processing. IAP handles different image sources and helps to organize phenotypic data by retaining the metadata from the input in the result data set. In order to measure phenotypic traits in new or modified setups, users can easily create new analysis pipelines or modify the predefined ones. IAP provides various user-friendly interfaces at different system levels to meet the demands of users (e.g. software developers, bioinformaticians, and biologists) with different experiences in software programming.  相似文献   
4.
The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.Introducing DNA-modifying enzymes rather than DNA-based expression cassettes is an attractive alternative for genetic engineering and genome-editing applications such as gene targeting or site-specific recombination. It offers a transient presence of the enzymes, and the process can be coordinated with high levels of enzymatic activity at the time and sites of the desired DNA recombination events. Many DNA-metabolizing enzymes (endonucleases, transposases, and topoisomerases), when delivered in an unrestrained manner, show adverse effects on cell viability. Delivery in the form of protein or RNA may help to mitigate these effects (Cui et al., 2011; Sander et al., 2011; Watanabe et al., 2012). In addition, by introducing proteins, one can avoid the need to remove the protein-encoding DNA fragments from the engineered plant genome. This may help shorten the time from laboratory to field for future improved germplasms.Site-specific recombinases such as Cre or FLP have been widely used in genetic engineering applications (Sorrell and Kolb, 2005). The 38-kD Cre enzyme specifically binds to and recombines the 34-bp loxP sequences, allowing the removal, integration, or inversion of the DNA fragment flanked by these sequences (for review, see Wang et al., 2011). There are a number of established methodologies designed to provide the Cre recombinase activity for site-specific recombination in eukaryotic cells that do not involve the delivery of DNA. These methods include lipofection (Baubonis and Sauer, 1993), microinjection of protein or mRNA (de Wit et al., 1998; Luckow et al., 2009), electroporation of protein or mRNA (Kolb and Siddell, 1996; Ponsaerts et al., 2004), or using modified microorganisms for Cre delivery to their host cells (Vergunst et al., 2000; Koshy et al., 2010). Another strategy that has been used is the incubation or injection of tissues/cell cultures with cell-permeant Cre, a modified Cre protein fused to protein transduction domains or cell-penetrating peptides (Jo et al., 2001; Will et al., 2002; Lin et al., 2004; Nolden et al., 2006).For biotechnological applications in plant sciences, protein delivery systems have been developed, including microinjection (Wymer et al., 2001), protein immobilization to gold particles (Wu et al., 2011), and protein transduction through cell-penetrating peptides (for review, see Chugh et al., 2010). The cell-penetrating peptides were shown to enable intracellular delivery of the Cre recombinase protein to rice (Oryza sativa) callus tissues (Cao et al., 2006). Nanobiotechnology is offering an attractive alternative, since nanoparticles can be precisely tailored to deliver a particular biomolecule to the cell, tissue, or organism of interest when needed (for review, see Du et al., 2012). Mesoporous silica nanoparticles (MSNs) are particularly suited for this purpose. These porous nanoparticles are formed by a matrix of well-ordered pores that confers high loading capacity of molecules like proteins (for review, see Popat et al., 2011). Additionally, surfaces of MSNs can be readily modified, permitting the customization of nanoparticles to particular experimental needs (for review, see Trewyn et al., 2007). In our previous studies, it was shown that MSNs can be used for the codelivery of DNA and chemicals (Torney et al., 2007) as well as DNA and proteins (Martin-Ortigosa et al., 2012a) to plant cells via biolistics. To improve MSN performance as a projectile, gold plating of MSN surfaces was performed, increasing nanoparticle density and, subsequently, the ability to pass through the plant cell wall upon bombardment (Martin-Ortigosa et al., 2012b).In this work, the Cre recombinase enzyme was loaded into the pores of gold-plated MSNs and delivered through the biolistic method to maize (Zea mays) cells containing loxP sites integrated into chromosomal DNA (Lox-corn; Fig. 1A). Lox-corn expressed the glyphosate acetyltransferase gene (gat) and the Anemonia majano cyan fluorescent protein gene (AmCyan1) flanked by loxP sites. The MSN-released Cre enzyme recombined the loxP sites, thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of a variant of Discosoma sp. red fluorescent protein gene (DsRed2) and the loss of the selectable marker gene (Fig. 1A). Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues, leading to successful genome editing.Open in a separate windowFigure 1.A, Schematic representation of the MSN-based bombardment technology. Cre protein is loaded into the pores of gold-plated MSN (Cre-6x-MSN) and subsequently bombarded onto immature embryos of a transgenic maize line carrying a loxP construct (Lox-corn). The parental transgenic Lox-corn tissues are blue fluorescence and herbicide resistant because they harbor a cassette with the glyphosate acetyltransferase (gat) selection gene and the AmCyan1 (cyan) marker gene flanked by the loxP sites. The DsRed2 (dsred) gene for the expression of a red fluorescent protein is placed downstream of the cassette. Once Cre recombinase is released inside the cell, it performs the recombination, excising gat-AmCyan1 genes and leading to the expression of the DsRed2 gene, switching the cell fluorescence pattern from blue to red. P, Promoter; T, terminator. UBINTRF, CYANF, and DSRED2R are primers for DNA analysis. B, Transmission electron microscope image showing the typical hexagonal shape and the well-ordered pore structure of a 6x-MSN. C, Scanning electron microscope image showing gold nanoparticle deposition (white dots) in all surfaces of 6x-MSN. D, Western blot showing Cre protein loading and release dynamics from 6x-MSN. The protein loading is almost immediate, even though some protein can be detected in the buffer even after 1 h of loading. For the release, some Cre protein can be observed after 24 h of incubation. Most of the protein remains in the 6x-MSN pellet. C+, 400 ng of Cre protein; Empty, a lane with no protein loading. The bands observed in the Empty lane were the spillover from the neighboring Pellet lane, which represents Cre-loaded 6x-MSN after the release experiment resuspended in Laemmli loading buffer (see “Materials and Methods”).  相似文献   
5.
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.  相似文献   
6.
The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.  相似文献   
7.
Toll-like receptors (TLRs) are best characterized for their roles in mediating dorsoventral patterning and the innate immune response. However, recent studies indicate that TLRs are also involved in regulating neuronal growth and development. Here, we demonstrate that the TLR Tollo positively regulates growth of the Drosophila melanogaster larval neuromuscular junction (NMJ). Tollo mutants exhibited NMJ undergrowth, whereas increased expression of Tollo led to NMJ overgrowth. Tollo expression in the motoneuron was both necessary and sufficient for regulating NMJ growth. Dominant genetic interactions together with altered levels of phosphorylated c-Jun N-terminal kinase (JNK) and puc-lacZ expression revealed that Tollo signals through the JNK pathway at the NMJ. Genetic interactions also revealed that the neurotrophin Spätzle3 (Spz3) is a likely Tollo ligand. Spz3 expression in muscle and proteolytic activation via the Easter protease was necessary and sufficient to promote NMJ growth. These results demonstrate the existence of a novel neurotrophin signaling pathway that is required for synaptic development in Drosophila.  相似文献   
8.
Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.  相似文献   
9.
In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifically, fibroblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK−/− mice) and their wild-type littermates (CERK+/+) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fibroblasts from CERK+/+ mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK−/− fibroblasts. This observed difference was reflected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the inflammatory stage to the peak of the fibroplasia stage (e.g., proliferation and migration of fibroblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fibroblasts isolated from CERK−/−. As the proper migration of fibroblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing response of wounds.  相似文献   
10.
Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.  相似文献   
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